Poster Session II and Coffee Break
(142) Long fragments achieve lower base quality in Illumina paired-end sequencing
Illumina's sequencing technology provides high quality reads of DNA fragments with error rates below 1/1000 per base. Runs typically generate millions of reads where the vast majority of the reads has also an average error rate below 1/1000. However, some paired-end sequencing data show the presence of a subpopulation of reads where the second read has lower average qualities. We demonstrate that the fragment length is a major driver of increased error rates in the R2 reads. Fragments above 500 nt tend to yield lower base qualities and higher error rates than shorter fragments. We show the fragment length dependency of the R2 read qualities using publicly available Illumina data generated by various library protocols, in different labs and using different sequencer models. Our finding extends the understanding of the Illumina read quality and has implications on error models for Illumina reads. It also sheds a light on the importance of the fragmentation during library preparation and the resulting fragment length distribution.
(116) Automated genomic DNA extraction from large volume whole blood
Whole blood samples are critical for disease prediction and diagnosis. The emerging field of personalized medicine has significantly increased the demand of researcher’s usage of large volume of blood specimens from Biobanks. Our goal was to provide a scalable genomic DNA (gDNA) extraction method from whole blood suitable for downstream applications such as qPCR, microarray analysis and next generation sequencing (NGS).
Here we present an automated, high-quality, research-ready DNA extraction from large volume blood from biobanks. GenFind V3 DNA Isolation Kit integrated on KingFisher Duo system, provides scalable and consistent system for gDNA extraction from 2 mL of whole blood. GenFind V3 DNA Isolation Kit uses SPRI (Solid Phase Reverse Immobilization) paramagnetic bead technology to isolate genomic DNA from fresh or frozen whole blood and serum containing Citrate, EDTA or Heparin anticoagulants. The combined automation and chemistry platform processing 24 samples within 2 hours. A comparison between manual extraction and automated extraction was done with multiple donors. Quality metrics for gDNA yield, purity and integrity were accessed by NanoDrop and gDNA TapeStation. The gDNA yield from 2 mL whole blood was between 40-80 µg. Recovered gDNA exhibited high purity with A260/A280 ratios greater than 1.8 and A260/A230 ratios about 1.6. DNA integrity as measured by DNA integrity number (DIN) was above 9.0. The extract gDNA using GenFind V3 and KingFisher Duo system was suitable for downstream applications such as PCR, Microarray and NGS.
(108) A 12 minute, single tube, nanogram input library prep
Next Generation Sequencing (NGS) library construction is a workflow bottleneck. The processes of library construction for DNA and RNA sequencing are complex, error prone, costly, and in general are time consuming. Current commercial products offer multistep, multi-hour sample processing that can be cost and labor prohibitive. These issues have created a demand for a simple, rapid, and cost-effective library construction product that offers users application flexibility while minimizing construction complexity for low to ultra-high throughput sample processing. SeqOnce has developed a novel library technology that is rapid and minimizes sample processing complexity. The five tube kit is comprised of formatted master mixes for a simple and stable user workflow. The 12 minute library construction uses a single master-mix that when combined with fragmentation and PCR steps produces libraries in less than 50 minutes. A single size selection step occurs after PCR and the PCR free protocol is less than 20 minutes. The kit contains three master mixes and includes adapters, which maximally promotes product flexibility across multiple applications.With master-mixes that are stable for 7 days at ambient temperatures, automation on liquid handling platforms is effortless due to the simple workflow. The technology has been validated with samples ranging from microbial to human and supports a wide range of nucleic acid types and input range: 1ng - 200ng. The sequencing data is either equivalent or superior to other competing library products, with high mapping rates and excellent performance across variable GC content. The technology is currently optimized for the Illumina platform and alternative NGS platforms are under evaluation for future development.
(174) Keanu: a novel visualization tool to explore biodiversity in metagenomes
One of the main challenges when analyzing complex metagenomics data is the fact that large amounts of information need to be presented in a comprehensive and easy-to-navigate way. Here, we describe development and application of a command-line tool for exploring sample content in metagenomics data. Keanu, a tool for exploring sequence content, allows a user to understand what organisms are present in a sample and their abundance by analyzing alignments against a BLAST database and displaying them in an interactive web page. The content of a sample can be presented as a collapsible tree, with node sizes indicating abundance, or a bilevel partition graph, with arc size indicating abundance. Here, we show how we used Keanu to explore shotgun metagenomics data from a sample collected from a bluff that contained paleosols in an alpine site in interior Alaska. The site contained a krotovina, which is an ancient nest of unknown origin. Keanu allowed us to explore the potential origin of the krotovina by visualizing the number of hits related to different taxonomic categories. In summary, Keanu provides a facile means by which researchers can explore and visualize species present in sequence data generated from complex communities and environments. Keanu is written in Python and is freely available at https://github.com/IGBB/keanu.
(160) Streamlined SMRTbell® library generation using addition-only, single tube strategy for all library types reduces time to results.
We’ve streamlined our SMRTbell library generation protocols with improved workflows to deliver seamless end-to-end solutions from sample to analysis. A key improvement is the development of a single-tube reaction strategy that shortened hands-on time needed to generate each SMRTbell library, reduced tedious AMPure purification steps, and minimized sample handling induced gDNA damage to retain integrity of the long-reads for sequencing. The improved protocols support all large-insert genomic libraries, multiplexed microbial genomes and amplicon sequencing. These advances enable completion of library preparation in less than a day with approximately 4 hours hands-on time and opens opportunities for automated library preparation for large-scale projects.
Here we share data summarizing performance of the new SMRTbell Express Template 2.0 Kit representing our solutions for >30 kb large-insert genomic libraries, complete microbial genome assemblies, and high-throughput amplicon sequencing. The improved throughput of the Sequel System with read lengths up to 30 kb and high consensus accuracy (>99.999% accuracy) makes sequencing with high-quality results increasingly accessible to the community.
(306) Flash Oxidation (FOX): A New and Improved Platform for Biopharmaceutical Hydroxyl Radical Protein Foot-printing Higher Order Structural Analysis
Hydroxyl Radical Protein Foot-printing (HRPF) is an emerging higher order structural (HOS) analysis technique. Several academic laboratories have demonstrated the utility of HRPF for HOS analysis, however, this technique has not been widely adopted by the research community. We have identified barriers that have limited the adoption of HRPF. These include 1) the use of dangerous and expensive lasers that demand substantial safety precautions; 2) the irreproducibility of HRPF caused by background scavenging of OH radicals.
Here we describe the development and performance of a Flash Oxidation System that replaces UV lasers with proprietary plasma lamp technology, creating a facile and safe bench-top means to perform HRPF HOS analysis. Apo-Myoglobin was labeled as was Angiotensin II. Singly, doubly, and triply oxidized products were produced. A Poisson distribution for each species was determined indicating the absence of artefactual changes in protein HOS highly consistent with excimer laser HRPF. Further, real-time measurement of effective hydroxyl radical yield provides reproducible labeling irrespective of background scavenging.
(300) ABRF-sPRG 2018-2019: Development and Characterization of a Stable-Isotope Labeled Phosphopeptide Standard
The mission of the ABRF Proteomics Standards Research Group (sPRG) is to identify and implement technical standards that reflect the ABRF’s commitment to accuracy, clarity, and consistency in the field of proteomics. There is broad interest in quantifying protein phosphorylation alterations in cellular signaling pathways under different conditions. The transient nature and low abundance of many phosphorylation sites makes this analysis challenging. Here we report on the follow up of the two-year sPRG study designed to target various issues encountered in phosphopeptide experiments. We have constructed a pool of heavy-labeled phosphopeptides that will enable core facilities to rapidly develop assays. Our pool contains over 150 phosphopeptides that have been previously observed in mass spectrometry data sets. The specific peptides have been chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling pathways: AMPK, death and apoptosis, ErbB, insulin/IGF-1, mTOR, PI3K/AKT, and stress (p38/SAPK/JNK). We feel this pool will enable researchers to test the effectiveness of their enrichment workflows and to provide a benchmark for a cross lab study. This standard should be helpful in number of ways, including providing a complete workflow solution for phosphopeptide enrichment, as an internal enrichment and chromatography calibrant, and as a pre-built biological assay for a wide variety of signaling pathways. Previously, we mixed the standard into an activated HeLa tryptic digest and distributed the mixture to over 60 ABRF member and nonmember laboratories around the world. We asked participants to enrich phosphopeptides out of the HeLa background and report ratios of the heavy phosphopeptides to the endogenous levels. In the current study, we continue validation of the standard within various RG group/ABRF members’ laboratories. The aim of this follow up study is to provide reagents, an optimized phosphopeptide enrichment protocol, instrument acquisition method parameters, and data analysis templates.
(166) Simultaneously DNA and RNA extraction from formalin-fixed paraffin embedded (FFPE) tissue
Formalin-fixed, paraffin-embedded (FFPE) tissues is an invaluable study resource for research, especially for biomarker detection. The advance of Next Generation Sequencing (NGS) allows researchers to study genomes, epigenomes and transcriptomes using limited sample material such as FFPE tissues. However, there are challenges for nucleic acids extraction from FFPE tissue due to how FFPE samples are prepared. The nucleic acid quality and integrity are often affected by duration of tissue fixation, age and storage condition of tissue blocks, and the extraction method.
Here we present a streamlined NGS ready to use sample prep workflow for FFPE tissue using a product in current development FormaPure XL Total (Beckman Coulter Life Science). FormaPure XL Total uses SPRI (Solid Phase Reverse Immobilization) paramagnetic beads to isolate DNA and RNA simultaneously. Up to 7 10 µm/curls of FFPE tissue can be processed by FormaPure XL Total in one single reaction. For researchers who have high throughput needs, we implemented an automated FFPE extraction method on a Biomek i5 Span–8 automated workstation using FormaPure XL Total. Total of 7 FFPE tissues that represent 4 different tumor types: liver, large intestine, breast and brain were demonstrated on a Biomek i5 Span–8 automated workstation. The work flow takes less than 6.5 hours with only 30 minutes hands on time. FormaPure XL Total provides consistent and efficient extraction performance from FFPE tissues, and it is easy to accommodate tissue input variations.
(178) MULTI-seq: Scalable Sample Multiplexing for Single-Cell RNA Sequencing using Lipid-Tagged Indices
MULTI-seq is rapid, modular, and universal scRNA-seq sample multiplexing strategy using lipid-tagged indices. MULTI-seq reagents can barcode any cell type from any species with an accessible plasma membrane in 10 minutes and also works for nuclei. The method is compatible with enzymatic tissue dissociation, and also preserves viability and endogenous gene expression patterns. We leverage these features to multiplex the analysis of multiple solid tissues comprising human and mouse cells isolated from patient-derived xenograft mouse models. We also utilize MULTI-seq's modular design to perform a 96-plex perturbation experiment with human mammary epithelial cells. MULTI-seq also enables robust doublet identification, which improves data quality and increases scRNA-seq cell throughput by minimizing the negative effects of Poisson loading. We anticipate that the sample throughput and reagent savings enabled by MULTI-seq will expand the purview of scRNA-seq and democratize the application of these technologies within the scientific community.
(314) Mass spectral Analysis of acetylated peptides: Implications in proteomics
Sequence determination of peptides using mass spectrometry plays a crucial role in the bottom-up approaches for the identification of proteins using different algorithms. Minimizing false discoveries or validating the sequence of the peptides is relevant for identification of proteins. Chemical modification of peptides followed by mass spectrometry is an option for improving the spectral quality. In silico derived tryptic peptides with different N-terminal aminoacids were designed from human proteins and synthesized. The effect of acetylation on the fragmentation in the CID mode was studied. N-terminal acetylation of the tryptic peptides was shown to form b1-ion, improve the abundance and occurrence of b-ions. In some cases, the intensity and occurrence of some y-ions also varied. Thus, it was demonstrated that acetylation plays an important role in improving the de novo sequencing efficiency of the peptides. The acetylation was extended to the tryptic peptides generated from the proteome of an Antarctic bacterium Pseudomonas syringae Lz 4W using the proteomics work flow and the fragments generated from the peptides were analysed. A total of 1,070 Proteins were identified from P. syringae Lz4W. A comparison of the MS/MS spectra of the acetylated and unacetylated peptides revealed that acetylation helped in improving the spectral quality and validated the peptide sequences. This method is also useful in detecting the N-terminal residue of the peptide. Using this method 673 of proteins were validated from the P. syringae Lz4W.
(130) Evaluation of commercially available 16S metagenomics kits
Metagenomics analysis has demonstrated to be a powerful tool for the study of niche-specific microbial communities with the purpose of elucidating their genetic diversity, population structure and impact on the environment in which they thrive. Although decreasing sequencing costs and new bioinformatics algorithms are making shotgun metagenomics a viable option, locus-specific approaches (e.g., 16S, ITS, etc.) are expected to remain very popular because of their low cost and ease of data analysis. As such, it seems appropriate that biotechnology companies would find it useful to exploit their position in the reagent market and offer user-friendly kits for effective and successful execution of metagenomics studies. Recently, several companies, including QIAGEN, Zymo Research and Swift Biosciences have begun to offer "everything included" kits for generating bacterial metagenomics libraries that can be sequenced on Illumina instruments. In this study, we sought to evaluate and compare the Zymo Research, Swift Biosciences and PacBio 16S analysis protocols. The Zymo kit uses two-primer sets to amplify V1 and V2, or V3 and V4. The Swift kit utilizes seven-primer sets to amplify and create libraries for all V1-V9 and fungal ITS1 and ITS2 genes in a single primer pool. In contrast, the PacBio protocol generates sequencing libraries for the full length of the 16S gene. Libraries were constructed by the three methods both on "mock" and "real-world" samples. The results and protocols are discussed in terms of the ease of use, cost and data quality.
(322) MetaboQuest: Tool for Metabolite Identification
In a typical untargeted metabolomic analysis of human samples by liquid chromatography-mass spectrometry (LC-MS), about 70% of the detected ions represent unknown analytes. While elucidation of the analytes with no putative metabolite identifications (IDs) remains a significant challenge, we have the opportunity to identify known-unknowns by ranking putative metabolite IDs using information derived from multiple resources. Untargeted metabolomic studies that use tandem MS (MS/MS) allow more accurate identification, if matching experimental MS/MS data can be found in spectral libraries. However, existing spectral libraries cover only a small fraction of known compounds. Thus, computational tools are highly desired to identify and score putative metabolite IDs by combining information obtained from MS/MS data and mass of the associated parent ion. We developed MetaboQuest, a browser-friendly cloud-based tool for identification and ranking putative metabolite IDs using compound databases, pathways, biochemical networks, and spectral libraries. The tool enables: (1) mass-based search for putative IDs against multiple databases; (2) score calculation based on isotopic pattern analysis; (3) putative ID ranking using network-based approach that exploits the inter-dependent relationships between metabolites in biological organisms based on knowledge derived from pathways and biochemical networks; and (4) spectral matching of MS/MS data against experimental and in-silico fragments, We used state-of-the-art libraries to implement MetaboQuest’s graphical user interface (GUI), which allow users to import m/z, RT, and MS/MS data and to export putative IDs in their desired format. Furthermore, the GUI provides users with interactive visualization of putative IDs, networks, and MS/MS spectra. The performance of MetaboQuest in terms of annotation accuracy and computational efficiency is compared against other existing tools using LC-MS/MS data from multiple metabolomic studies. We believe MetaboQuest will contribute to addressing the major bottleneck in metabolomics - metabolite identification, thereby enhancing the contribution of metabolomics in studies such as biomarker discovery and systems biology research.
(316) Purification and Characterization of Proteins and Peptides from Marshmallow (Althaea officinalis) Seeds
Marshmallow (Althaea officinalis) belongs to family Malvaceae, is a medicinal plant used in traditional and alternative medicine in various parts of the world. Marshmallow is one of the seven medicinal plants that are collectively known as Joshanda. It is herbal preparation which has been used traditionally as a home remedy for the treatment of common cold, coughs, nasal congestion, and fever. Previous studies showed that crude extract and/or purified compounds from flowers, root and leaves from Marshmallowhave have anti-microbial, anti-inflammatory, and many other pharmacological effects. However, there is limited data reported about the structure and functions of proteins isolated from the seeds. Therefore, this study is carried out to purify and characterize proteins and peptides from Marshmallow seeds as well as to evaluate their antimicrobial activity against Salmonellaenterica, Staphylococcus aureus, Saccharomyces cerevisiae and cytotoxic activities in MCF-7 breast cancer cell line. Extraction of proteins was performed in 20 mM Tris/HCl pH 8.0, then purified by the combination gel filtration and reverse phase HPLC. Purified proteins were analyzed by SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometer. The partial amino acid sequence of 22kDa protein was established up to 25 residues by N-terminal amino acid sequencing using protein sequencer (PPSQ Shimadzu). The data suggest that Marshmallow RP-HPLC purified protein is a monomeric protein of 22kDa. The amino acid sequence alignment using BLASTp revealed similarity in 22/25 amino acid residues with Thaumatin from Corchorus capsularis. The MTT assay of Marshmallow crude protein led to dose dependent cytotoxic effect in MCF-7 cells with an IC50 value of 163.29 µg/ml. The gel filtration pooled fractions 5-8, 9-11, and 12-13 showed 58%, 46% and 32% inhibition respectively. The antimicrobial activity of crude protein extract showed 69% inhibitory activity against Staphylococcus aureus.
(114) An Unbiased Comparison of BigDye Terminator and BrilliantDye Terminator
Dye terminator cycle sequencing utilizes both unlabeled dNTPs and fluorescently labeled ddNTPS to PCR amplify DNA templates. In addition to labeling the DNA fragments, the modified ddNTPs also terminate the PCR extension, generating DNA fragments of varying sizes. Fragments are separated by capillary electrophoresis, during which laser excitation enables identification of each individual base. Dye terminator reaction kits are available from many scientific vendors. Here we compare Applied Biosystem’s (ABI) BigDye Terminator v3.1 cycle sequencing kit to Edge Biosystem’s BrilliantDye Terminator v3.1 cycle sequencing kit. A sample of BrilliantDye Terminator was received from Edge Biosystem for testing purposes. Both reagents were diluted, per our protocol, 1:128 or 1:10 depending on sample type. We sequenced 300 plasmid and PCR products in duplicate. Initial findings showed little to no difference between the two reagents for sequences shorter than 600bp. In sequences longer than 600bp, we noticed an increase in the length of high quality reads with the BrilliantDye Terminator compared to BigDye Terminator, achieving around 50-100 additional high quality, readable bases. We also saw that in some of the longer reads, there was a decrease in signal strength, but the raw data was more uniform and the traces were clearer when using BrilliantDye. Overall, in our opinion, the BrilliantDye Terminator and BigDye Terminator performed similarly for sequence reads up to 600bp, but the BrilliantDye outperformed BigDye in sequences greater than 600bp.
(112) A Robust, Streamlined, Enzyme-based DNA Library Preparation Method Amenable to a Wide Range of DNA Inputs
Advances in next generation sequencing (NGS) platforms have outpaced those in library preparation. While hundreds to thousands of NGS libraries can be sequenced in a single run of an Illumina instrument, a single library is constructed using a multi-step procedure requiring expensive consumables and specialized equipment. To overcome these limitations and increase the ease and throughput of library construction, we have developed a robust, streamlined library construction method that integrates enzyme-based DNA fragmentation with end repair and dA-tailing. The NEBNext Ultra II FS DNA Library Prep Kit eliminates the need for specialized equipment to fragment DNA and reduces the number of steps in the library construction protocol. This method produces high quality libraries from gDNA isolated from organisms whose genomes vary widely in GC content, as well as amplicons and DNA purified from blood. In addition, the FS kit generates libraries with substantially higher yields than those using mechanically sheared DNA, enabling greatly reduced input requirements. Libraries constructed using the FS kit from inputs as low as 100 pg of human gDNA for amplified libraries and 100 ng for PCR free, show similar coverage uniformity and GC bias compared to libraries constructed with 100 ng of mechanically sheared DNA.
The ability to generate high quality libraries from low amounts of starting material and a broad range of inputs will help advance the widespread implementation of NGS in both basic science and the clinic.
(208) Neuroprotective roles of fractalkine in multiple sclerosis: Characterization of novel humanized animal model
Multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS is the leading cause of nontraumatic neurological disability in young adults. Immune mediated destruction of the myelin and oligodendrocytes are considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability.
In an effort to understand microglia function during CNS inflammation, our laboratory showed that Fractalkine/CX3CR1 signaling regulates microglia neurotoxicity in models of neurodegeneration.
Fractalkine (FKN), a transmembrane chemokine expressed in the CNS by neurons signals through its unique receptor, CX3CR1 present in microglia. During EAE, CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ~20% of the population, exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and effect on neurons during MS remains unsolved.
The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses in absence of CX3CR1 signaling enhance neuronal/axonal damage.
We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibit exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and CNTF during EAE as WT mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation.
(144) Miniaturized automated PCR setup
Traditionally, PCR has been carried out in total reaction volumes of 20 – 50 μL. Reducing reagent volumes has the potential to reduce reagent consumption and cost. However, in practice the potential for miniaturization is often limited by the inability of conventional liquid handling methods to accurately transfer the required low volumes. High viscosity, e.g. of enzyme mixes or genomic DNA samples, is a particularly difficult challenge for conventional liquid handling.
Here we demonstrate the automation and miniaturization of genomic PCR setup using the mosquito® genomics pipetting robots. Using positive displacement pipetting, these instruments handle liquids of a wide range of viscosities with high precision and accuracy in the microliter-to-nanoliter range.
End-point PCR from human genomic DNA at a conventional 40 µL volume with manual set-up was compared with 10 µL, 5 µL or 2 µL reaction volumes and automated set-up.
Using only 2 µL total reaction volume, the potential for additional reagent and material savings was explored by further reducing template DNA and primer quantities.
We show that end-point PCR reactions can be readily miniaturized to as little as 2 μl total volume, while maintaining sensitivity and specificity
(150) NGS DreamPrepTM: A fully automated NGS library preparation solution from sample to quantified, pooled, and sequence-ready libraries
Illumina NovaSeq S4 flow cells have the ability to generate 10 billion reads in a single run. To maximize this level of throughput and achieve the optimal cost savings, hundreds of samples may be pooled for each run. Manual library preparation of hundreds of samples can result in long turnaround times, increased errors, poor reproducibility and may require multiple lab personnel.
The NGS DreamPrep is a turnkey solution designed and optimized for completely automated NGS library preparation and quantification. It combines Tecan’s library preparation reagents, automation and fluorescence plate readers to produce sequence-ready, normalized and pooled libraries. Innovative library preparation technology minimizes workflow time and user interaction during automation. Integration of NuQuant, a novel library quantification technology, into the library preparation allows automated library quantification, normalization and pooling on the instrument with no sample loss to QC procedures. The NGS DreamPrep system includes a Fluent liquid handling platform optimized for NGS library preparation with integrated Infinite F Nano+ plate reader for fast library QC.
The NGS DreamPrep enables flexible library preparation for up to 96 samples in less than 4 hours for DNA and approximately 8 hours for RNA. Preparing 96 DNA samples using identical inputs generated libraries with no adaptor dimers, no dropouts and no edge effects. The mean yield (as measured by NuQuant) of all 96 libraries was 249.3 nM with a standard deviation of 24.6 nM showing high sample-to-sample reproducibility across the entire plate. Library molar concentrations were quantified by the integrated plate reader and subsequently pooled. Sequencing results showed a %CV of <15% between the number of sequence reads, demonstrating the accuracy of NuQuant library quantification. The NGS DreamPrep is an innovative solution for high-throughput NGS library preparation and quantification with walk-away automation.
(148) Multi-site Evaluation of Next-Generation Sequencing Methods for Small RNA Quantification
SmallRNAs (smRNA) have been identified as important regulators of many processes in the cellular life cycle, from embryonic development, to cell differentiation, growth and proliferation, to cell death and apoptosis. There are several currently available technologies to assay the small RNA fraction from biological samples. While most technologies such as microarrays, RT-qPCR, and Northern Blot are probe-based methods, only massively parallel next generation sequencing (NGS) allows for discovery of new miRNAs. Widespread access to NGS instrumentation through research centers and core facilities coupled with decreasing sequencing costs has made NGS an increasingly attractive option for small RNA analysis. While standard RNAseq library preparation has become routine in most sequencing facilities, small RNA sequencing library preparation has historically been challenging due to high input amount requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are non-linear and do not reflect the accurate levels of small RNAs in vivo. Recently, a number of new kits have become available that permit lower input amount along with less laborious gel-free protocol options. Many also claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to drastically deplete adapter-dimer contamination in the resulting libraries. With the increasing number of smallRNA kits available, understanding the relative strengths of each method is critical for appropriate experimental design. In this study, we systematically compared eleven commercially available small RNA library preparation kits as well as Nanostring probe hybridization across multiple study sites. Kits were tested to evaluate diversity of library composition, non-linearity of detection, and ease of use.
(176) Automated library preparation for the MCI Advantage Cancer Panel at Miami Cancer Institute utilizing the Beckman Coulter Biomek i5 Span-8 liquid handler
Introduction: MCI Advantage Panel is a next generation sequencing (NGS) assay based on TruSight Tumor 170 (Illumina). The panel is designed to detect genetic alterations [e.g. single nucleotide variants (SNVs), insertions and deletions (indels), amplifications (CNVs), and fusions/splice-variants (SVs)] in 170 cancer associated genes.
Methods: Reference materials and patient samples (N=87) were utilized to establish the assay’s performance characteristics. DNA/RNA extractions were performed from formalin fixed paraffin embedded (FFPE) samples using FormaPure Total (Beckman). Libraries were constructed with TruSight Tumor 170 kit on the Biomek i5 Span-8 NGS Workstation (Beckman). Sequencing is performed using Illumina NextSeq550 sequencers. Bioinformatic analysis is performed with the TST170 apps and Variant Interpreter on the HIPAA-compliant BaseSpace Enterprise platform. The sequence data were also analyzed and reports were generated with the Philips IntelliSpace Genomics platform.
Results: Of the 108 DNA libraries processed, exon coverage at 100X or greater was very high across all DNA libraries (mean 99.78% with a standard deviation of 0.09%). RNA sequencing metrics show that for the 80 samples sequenced median insert length varied somewhat more than the DNA libraries (mean 109bp with a standard deviation of 14bp). RNA Median CV Coverage at 1,000X was consistent (mean 0.53 with a standard deviation of 0.09). Analysis of 77 clinical FFPE samples showed 98.7% concordance with the orthogonal methods.
Conclusion: Targeted NGS panel brings a unique opportunity for detection of multiple somatic alterations with a single platform in community cancer center setting. MCI Advantage Panel has an excellent overall coverage and shows high concordance with orthogonal methods.
(312) Let it brie: Using the non-destructive SepQuant dropletProbe sampling system to analyze the free amino acid content of various cheeses
Casein proteins are an important energy source for the microbial communities that drive the complex process of surface-ripening of various types of cheese. The process of proteolysis involves cleavage of these caseins into smaller peptides, then free amino acids, and culminates in amino acid catabolism, which is one of the primary biochemical reactions that produces flavor in cheese. Here, we aimed use the SepQuant dropletProbe, an innovative liquid extraction surface analysis sampling system, coupled to high performance liquid chromatography-mass spectrometry (HPLC-MS) to profile the free amino acid content of 12 different cheeses. Using this approach, we were able to detect 17 separate amino acids across 37 samples, as well as to compare and contrast the free amino acid profiles within and between samples. The ability of the SepQuant dropletProbe to automate extraction and to accommodate samples of varying heights eliminated extensive sample preparation steps that are usually required for this type of biochemical analysis. Results indicated that the relative quantity of free amino acids varies in association with cheese hardness and between the rind and body sections of cheeses. Unique to this study, we were also able to observe that the relative quantity of amino acids varies within the sample (e.g. between the inner and outer parts of the rind). In this study, we demonstrated that the SepQuant dropletProbe sampling system is a sensitive, efficient, and non-destructive method to characterize the free amino acid content of many different types of cheese. Future research could aim to apply this same concept to a variety of different known flavor compounds in cheese and other foodstuffs and to monitor the production of flavor compounds from the same sample in situ over the course of time.
(128) Evaluating Next Generation Sequencing library quantification methods
Accurate library quantification is a critical step in Next Generation Sequencing (NGS) application quality control. Library concentration, taken together with size, are used to load equimolar ratios of libraries onto the sequencer, ensuring optimal cluster density and maximizing data output. Quantitative PCR (qPCR) is the gold standard for determining the concentration of library template molecules that contain both of the adapters necessary to bind to the flow cell and generate clusters. Other methods such as fluorometric quantification, digital PCR, or bead based normalization are also used, depending on library type and the needs of the user. Here we compare library quantification by qPCR to library quantification using a fluorometric assay in an effort to provide cost savings to our customers. We measured the concentration of several library types side by side using both qPCR and fluorometric assays. Both ChIP-seq and ATAC-seq library concentrations were higher by fluorometric assay than qPCR, while mRNA-seq library concentrations were lower. We then pooled libraries based on the molar concentration as determined by each method, sequenced the pools, and evaluated the cluster density and library distribution of each pool. The percentage of each library was different depending on whether it was quantified by fluorometric assay or qPCR, but the overall distribution of libraries was generally similar between methods. In conclusion, qPCR is the preferred method for library quantification, but it may be possible to quantify using a fluorometric assay for libraries of similar type, size, and quality.
(122) Core Analysis Viewer: Simplifying Omics Data Delivery, Training, and Interpretation
In recent years, genomic and other data intensive approaches have become increasingly common in the life sciences. The decreasing costs of sequencing have made such methods approachable for a wide range of researchers, many with limited knowledge of how to analyze and interpret such data. The Bioinformatics Core Facility at the University of Delaware provides our users with analysis pipelines providing primary bioinformatic analysis of many omics data types including RNA-Seq, ChIP-Seq, SNP detection, and 16S rRNA analysis, as well as downstream pathway and network analysis. While such support from a core facility can take care of the “computational heavy-lifting” associated with such projects, the key data interpretation portion of such projects is most often a task that requires the client’s knowledge of their system. We have seen that many clients find themselves ill-prepared to manage the plethora of reports and tables that can result from primary analysis, and thus have great difficulty in effectively interpreting their data. To standardize and simplify this task we have built a Core Analysis Viewer (CAV) web application. This application provides a web-based framework for presenting analysis results in a user-friendly environment. The framework can be adapted to accept data from various analysis pipelines. For instance our RNA-Seq CAV viewer provides the user with details and summary graphics of their data through all steps of analysis from raw data, to QC/trimming, reference mapping, determination of gene and exon counts, and differential expression analysis. By providing results in a user-friendly and consistent web-environment, online training modules can be deployed that walk users through the various metrics and results they can expect from an analysis, and aid them in building skills needed to interpret the data.
(206) Integration and validation of RNA ISH, multispectral imaging and analysis protocols into a Core environment
Identification of biomarkers is a major goal of personalized medicine. Large transcriptome screens have identified new targets and molecular signatures for disease sub-types. However, tissue spatial information, which fundamentally alters in vivo cell behavior and gene expression, is lost. To understand spatial context and validate bulk tissue screens, most researchers rely exclusively on antibodies and immunostaining assays. Unfortunately, this is either not the appropriate choice for some targets, such as long non-coding RNAs, or it is not feasible because no reliable antibodies exist. To address these issues, we have established an alternative work-flow incorporating RNA in situ hybridization (CISH and FISH), whole slide and/or multispectral scanning and image analysis.
Methods: RNAscope® technology; Leica SCN scanner or Vectra3 multispectral imaging system for image acquisition; , ImageJ2/FIJI, R, InForm and Visiopharm software platforms for quantitative analysis.
Results: Several laboratories have used this workflow to address their specific questions. For example, we established and validated RNAscope assays for signaling factor transcripts, which are now integrated into an ongoing clinical trial. In this case, all tested commercial antibodies failed the validation assay. We also assessed expression of LNC RNAs in prostate cancer and put in place protocols for normalizing probe quantification across samples. The analysis revealed that storage and/or sample preparation affected the detection of certain LNC RNAs more than others identifying important factors regarding banking specimens. Spatial heat map visualization of RNAscope probes revealed an unexpected distribution of inflammatory cytokine targets in the kidney which are now being further investigated. In conclusion, RNA ISH is a powerful alternative strategy for assessing the spatial distribution of specific cell populations and critical biomarkers within intact tissues. This approach coupled with sophisticated imaging modalities and downstream analysis support provides new collaborative opportunities for Core laboratories.
(120) Comprehensive Detection of Germline and Somatic Structural Mutation in Cancer Genomes by Bionano Genomics Optical Mapping
In cancer genetics, the ability to identify constitutive and low-allelic fraction structural variants (SVs) is crucial. Conventional karyotype and cytogenetics approaches are manually intensive. Microarrays and short-read sequencing cannot detect calls in segmental duplications and repeats, often miss balanced variants, and have trouble finding low-frequency mutations.
We describe the use of Bionano Genomics’s Saphyr platform to comprehensively identify SVs for studying cancer genomes. DNA >100 kbp is extracted, labelled at specific motifs, and linearized through NanoChannel arrays for visualization. Molecule images are digitized and de novo assembled, creating chromosomal arm scale genome maps. Somatic mutations can be identified by running the variant annotation pipeline that compares the cancer sample’s assembly SVs against >600,000 SVs in Bionano’s control sample SV database, and against a matched control sample’s SVs, if avaliable. Also, two new Bionano pipelines leverage these long molecules to identify additional somatic SVs: the copy number variation (CNV) and the molecule mapping pipelines. By examining the coverage-depth of molecules alignment to the public reference, the pipeline can identify megabases long CNVs. Similarly, clusters of split-molecule alignments can reliably find translocations and other rearrangements.
We applied this suite of discovery tools to identify SVs in a well-studied melanoma cell line COLO829. We collected data from the tumor and the matched blood cell line, constructed contiguous assemblies (N50 >50 Mbp), and called >6,000 SVs in each genome. Then, we classified 51 as somatic by comparing the tumor and the blood control. The two new pipelines further increased sensitivity to rearrangements, for example they captured a BRAF duplication, and other chromosome-arm CNVs. We apply these thorough approaches to multiple well-studied cancer lines to identify novel SVs missed by previous studies. In conclusion, with one comprehensive platform, Saphyr can discover a broad range of traditionally refractory but relevant SVs, and further improves our understanding of cancer.
(136) High-Throughput, Low Volume gDNA Extraction from Whole Blood Enabled by Covaris Adaptive Focused Acoustics (AFA) and oneTUBE-10
Extraction of genomic DNA (gDNA) from fresh whole blood is the first step in multiple translational research and molecular diagnostics applications, such as next-generation sequencing (NGS), multiplex PCR, qPCR, and droplet digital PCR (ddPCR). For example, DNA from whole blood is used as a matched-control for solid tumor somatic mutation profiling and for the detection of clinically-relevant variants in hematological malignancies, such as leukemia and myelodysplastic syndrome. An increasing number of clinical studies demonstrate the value of detecting disease-specific biomarkers from blood for early detection, diagnosis, monitoring treatment efficacy, and cohort study recruitment. Scaling down fresh blood volumes while scaling up processing capabilities is desirable to maximize laboratory throughput. At present, most DNA extraction methods require high volumes of blood and are challenging to automate because centrifugation or vacuum equipment are necessary. Additionally, conventional column and magnetic-based workflows need larger volumes for wash and elution steps, which require the use of deep well plates and other specialized consumables. Larger volumes are also needed to avoid excessive viscosity of the lysate, which can interfere with magnetic bead separation. To circumvent sample and process-related challenges, Covaris has adapted the oneTUBE-10 Plate for streamlining high-throughput nucleic acid extractions from whole blood using Adaptive Focused Acoustics (AFA) technology. Here, we performed 12 DNA extractions from healthy donors to evaluate recoveries, sample quality, and purity. Our results show recoveries in the range between 376 to 809 ng with an average fragment size of >1 kb. We also show that the extracted and purified DNA is devoid of any detectable PCR inhibiting contaminants. Taken together, this less than 90-minute AFA-enabled workflow significantly improves blood cell lysis and reduces hands-on time. The workflow can be performed on a liquid handler using the integrated Covaris R230 Focused-ultrasonicator, or off-deck using the LE220-plus Focused-ultrasonicator.
(200) Automated single cell tracking of in vitro multi-cellular morphogenesis predicts pluripotent stem cell fate decisions
During stem cell differentiation, individual cells must undergo a series of lineage commitment decisions to reach their final cell fate, analogous to the transitions that occur during early development. While many transcriptional and microscopic techniques exist to assess the quality of differentiation from fixed samples, there are few approaches capable of providing non-destructive monitoring of stem cell cultures. To enable live assessment of in-vitro differentiation, we have developed an assay to detect changes in human pluripotent stem cell behavior by imaging nuclear labeled cells using confocal microscopy. By developing a cell tracking algorithm based on an ensemble of neural nets, we are able to detect changes to cell and colony parameters such as migration velocity, cell density, and persistence of migration at single cell resolution. Using this platform, we explored changes to cell behavior during the early stages of common differentiation protocols to induce multilineage (using BMP4) or directed differentiation (using CHIR or dual SMAD inhibition). We show that single cell tracking behavior assessment strongly correlates with canonical loss of pluripotency (OCT4 and SOX2) and acquisition of early lineage markers (EOMES). Furthermore, whole colony analysis reveals regions where cell behavior changes in advance of canonical lineage marker expression, enabling prediction of localized differentiation within the larger stem cell population. These results indicate that assessing single cell behavior through whole colony tracking provides a rapid and effective tool for non-destructively assaying pluripotent stem cell fate decisions.
(404) Research Resource Identifiers (RRID) for Core Facilities and Research Equipment
The reproducibility of research results is one of the key tenets of scientific discovery. These results are often generated using equipment located in a scientific research laboratory. Thus, it would stand to reason that sufficient, detailed, and transparent reporting of equipment is key to allowing researchers to assess the validity of previous findings. The cost of irreproducible studies has been well documented and the lack of specific and unambiguous reference to research tools contributes significantly to this cost. The scientific community currently lacks a structured citation style or method for tracking what types of scientific lab equipment are being utilized to conduct research on grant funded projects or peer reviewed publications. In turn, this makes it difficult for researchers to reproduce the results of other researchers and thus, contributes to the reproducibility crisis the scientific community is facing. To combat this problem, a team of librarians and scientific researchers at Florida State University and the University of California-San Diego are developing a tool that will provide a structured citation style for scientific lab equipment. The name of this tool is the Universal Scientific Equipment Discovery Tool (USEDiT). The poster will focus on our efforts to develop a novel citation index for scientific equipment, that will make unambiguous citing of equipment as easy as it is to cite authors in a study.
(110) A Low DNA Input Protocol for High-quality PacBio De Novo Genome Assemblies
A high-quality reference genome is an essential tool for studying the genetics of traits and disease, organismal, comparative and conservation biology, and population genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that may have lower DNA content or on projects with limited input DNA for other reasons.
Here we present a modified SMRTbell library construction protocol without DNA shearing or size selection that can be used to generate a SMRTbell library from just 150 ng of starting genomic DNA. Remarkably, the protocol enables high quality de novo assemblies from single invertebrate individuals and is applied to taxonomically diverse samples. By sequencing and assembling material from a single diploid individual, only two haplotypes are present, simplifying the assembly process compared to samples from multiple pooled individuals.
The libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating ~11 Gb of sequence per SMRT Cell with 10 hour movies, and followed by de novo genome assembly with FALCON. The resulting assemblies had high contiguity (contig N50s over 1 Mb) and completeness (as determined by conserved BUSCO gene analysis) when at least 30-fold unique molecular coverage is obtained.
This new low-input approach now puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life. The method presented here is scalable and can be applied to samples with starting DNA amounts of 150 ng per 500 Mb genome size.
(158) Sequencing complex genomes with PromethION technology in a core setting
Many genomes are characterized by rearrangements confering novel phenotypes; including drug resistance, growth rate, desirable agricultural traits, and disease progression. Advances in long-read sequencing have shown that short-read approaches miss a many large, structural events. Until recently long-read sequencing has been limited in scope due the high costs and high input material requirements. The Oxford Nanopore PromethION offers a lower cost and lower starting material alterative to other long-read sequencing platforms. These changes will make long-read sequencing a more routine technology in genomic studies. At this time, CSHL has developed pipelines to rapidly sequence plant and human samples. To date we have completed >5 high coverage human genomes with per cell yield ~70Gb, and 100 plant genomes with yields ~90Gb. Our top yield has been >142Gb on a single cell. This work has allowed an in depth exploration of the structural events in cancer progression and plant breading characteristics.
Despite these successes, integrating a PromethION in to core setting is a challenging prospect . Unlike other technologies, the system is not as robust to adjuncts that may be carried over from DNA extractions. This has the potential to require significant troubleshooting efforts that must be accounted for in the time and costs associated with projects. This is further compounded by variations in the flowcells themselves leading to uncertainly in the total data generated for a project. Another factor that much be considered is data management; as ~2Tb of data can be generated per flowcell. Basic data management methods like basecalling and data transmission can quickly overwhelm many data management systems. At this time CSHL is making plans for enhanced data storage and analysis systems to better enable large scale PromethION studies. This presentation will outline CSHL’s experience with sequencing on the Oxford Nanopore PromethION and recommendations on best practices for a core facility.
(402) Delivering greater value by operating core facilities under a quality management system.
The term ‘high value, low wastage’ research, ‘reproducibility crisis’ or more recently the ‘Innovation Opportunity’ have been topics of recent discussion in the research community (Australia’s National Health and Medical Research Council’s Research Translation Symposium). These discussions, initially stimulated by the 2012 published Nature article by Begley and Ellis citing Amgen could not confirm research findings of nearly 90% of 53 high profile papers in preclinical cancer studies. All academic institutions want to ensure they are producing high quality research and excellence, but also in challenging funding times, need to ensure they are optimising the use of resources. In 2014, Monash University had an ambitious vision to address this conversation, by implementing an international recognised quality management system across all research infrastructure core facilities. Fast forward to 2019, all 30 core facilities (35 sites) are certified to the ISO9001:2015 management standard and operate at best practice, with a commitment to deliver value through aligning with quality goals and fulfilling needs and expectation of users and key stakeholders.
Monash University’s research agenda is underpinned by an integrated network of world-class research infrastructure and by implementing ISO9001 over the past five years, we have been able to demonstrate strategic alignment, optimisation of resources, the ability to champion long-term success and overall excellence of the University. A recent independent review of the Monash Technology Research Platforms (MTRP) identified a number of critical success factors that the platforms contribute to and by using a strategic planning diagnostic tool and other feedback mechanisms as defined in the Platform Quality Management System (PQMS), we have been able to show overall strategic alignment, the ability to fulfil user expectations and reach quality goals, increase efficiency gains and provide a mechanism to address issues and continual improvements.
(126) Echo® System-Enhanced SMART-Seq v2 for RNASeq Applications
As next-generation sequencing costs have continued to decline by orders of magnitude in the past ten years, researchers are enabled to ask more questions about the transcriptome, with greater degrees of complexity. Oncologists are tracking RNA as cancer biomarkers, synthetic biologists assess transcription levels in designer pathways, and single-cell researchers are comparing individual cell expression to populations. Consequently, the variety, quantity, and demands of RNA sequencing experiments have all increased. SMART-Seq v2, a method developed by Simone Picelli et al at the Karolinska Institute, has become a ubiquitous method for single-cell and population RNA sequencing. Here, we demonstrate miniaturization of this process on human brain total RNA by utilizing the Echo 525 Liquid Handler. We show that this enhanced process can effectively reduce reagent cost for the workflow, and see that genes are not differentially expressed across miniaturization and input variables via the Illumina RNAExpress analysis in BaseSpace. Across all our conditions, we see an average transcript total alignment to reference of 94% with a 1. 6% CV. Thus, we provide a platform to address current throughput and affordability needs of RNA sequencing.
(154) Reproducibility of indel formation rates by comparing guideRNA format and delivery method
Multiple configurations of guideRNA and Cas9 components can be used for editing cells. A few options include: a plasmid expressing both the guideRNA and Cas9, Cas9 protein combined with a synthetic single guideRNA, and Cas9 combined with a synthetic 2-part guideRNA. In addition, delivering the components to cells can be done using lipofection or nucleofection transfection methods. In the GERG 2017 survey (https://abrf.org/sites/default/files/gerg_survey_poster_2018_final_0.pdf), plasmid format and lipofection delivery were favored among cell culture users. Meanwhile, RNP format for the guideRNA and Cas9 is gaining in popularity in combination with nucleofection delivery. This study aims to compare cutting efficiency at 3 different guideRNA targets based on the guideRNA format and delivery method across multiple labs. Determining which method or format is the most reproducible will be beneficial. Core facilities or research labs getting started with genome editing could use these results as a benchmark for optimizing their own protocols.
(318) Total Solubilization of FFPE Samples for High-Throughput Clinical Proteomics in One Step
Formalin fixed paraffin embedding (FFPE) is a decades-old sample preparation technique common in experimental research and medicine. FFPE embedded samples can be stored indefinitely at room temperature, resulting in an exceptionally large and rich worldwide collection. Despite its potential, proteomic analysis of FFPE samples has lagged. Traditionally, samples are first laboriously deparaffinized with often-toxic organic solvents. Subsequent protein extraction is extremely critical but no consensus has been reached as to an optimal protocol. Standardization is fully lacking.
Here, we present a one-pot solution which eliminates deparaffinization and which employs 5% SDS and S-Trap sample processing to exhaustively solubilize entire FFPE samples evaluated on human liver. Samples were paired and split, half flash-frozen and half fixed in formalin and paraffin embedded according to standard histopathology procedures. All samples were extracted with SDS using standard techniques (pulverization, syringe needles), probe sonication or Covaris AFA ultrasonication. SDS was removed by standard precipitation or S-Traps. Protein identification rates and reproducibility were evaluated after analysis on a Thermo QE HF-X or Fusion mass spectrometer.
Compared to standard procedures, the use of S-Traps resulted in significant increases in peptide (≥30%) and protein identification rate (≥20% increase) with greater reproducibility. The use of AFA decreased hands-on time, increased ID rates an additional 6% – 8% and significantly increased protein yield from FFPE samples (80% – ≥200%). The combination of S-Traps and AFA yielded ID rates comparable to those obtained from fresh frozen tissue (101%/97% ID rates for peptides/proteins) and by eliminating deparaffinization, prevents exposure to toxic xylene while saving approximately 6 hrs in sample processing.
Our system solves the problem of extraction bias and achieves the goal of reproducible standardized protein recovery from FFPE samples in a workflow suited to automated, high-throughput analyses. We anticipate this workflow will assist to usher in a new era of clinical proteomics.
(164) Whole Genome Sequencing of Microbial Communities for Scaling Microbiome and Metagenomic Studies
Our understanding of the role of human microbiome in health and disease has been growing rapidly in recent years. Amplicon sequencing of highly conserved 16S ribosomal RNA (rRNA) regions has long been the standard technique used to assess patient microbial diversity, however there are limitations to this method. 16S rRNA amplicon sequencing only captures prokaryotic diversity and misses eukaryotic and viral components of the microbiome. While an additional amplicon sequencing of the internal transcribed spacer 1 (ITS1) region can capture fungal diversity, there is no known parallel technique for viral detection. Furthermore, these rRNA amplicon methods are generally only genus-specific. To obtain species-level differentiation, multiple variable regions of the rRNA need to be assessed in repeated experiments. Additionally, important strain information is not detected. Strain-to-strain variation is responsible for pathogenicity, toxins, virulence factors, epitopes, and antibiotic resistance characteristics.
Whole genome sequencing (WGS) can determine strain-to strain variation and has more applications beyond microbial community identification. Metagenomic homolog discovery from uncharacterized organisms is enabling pathway design for production of molecules of interest. Using WGS, biologists are prospecting for natural enzymatic solutions to their challenges. Miniaturizing WGS significantly lowers the cost to sequence and discover, as well as reduces process cycle time. As researchers continue to deepen our understanding of human microbiome, and as biologists explore the metagenomic space, our tools and analyses need to scale accordingly.
A workflow utilizing the Labcyte® Echo® 525 Liquid Handler can provide the solution to cost-effectively scale WGS of microbiomes to meet the demands of this new era of microbiome research. In this study, we demonstrate that the Echo 525 Liquid Handler can be used to miniaturize WGS of microbial communities. Raw data can be processed, analyzed, and ready for interpretation within the hour, by utilizing CosmosID’s best-in-class microbiome bioinformatics platform. We see accurate representation of microbiome samples to their references.
(124) Depletion of Abundant RNAs in Eukaryote, Blood Samples, and Bacteria Increases Sensitivity of Next Generation Sequencing-Based Transcriptome Profiling
A challenge in whole-transcriptome sequencing is the large dynamic range of transcript expression within a total RNA sample. Highly expressed transcripts with minimal biological interest can dominate readouts, masking detection of more informative lower abundant transcripts. Here, we present a method to enrich for RNAs of interest by eliminating abundant, typically unwanted, RNAs before sequencing. This method is based on hybridization of probes to the target RNA and subsequent enzymatic degradation of the bound RNAs and the probes.
We optimized this method to remove abundant RNAs (such as ribosomal RNA) from human, mouse, rat, and bacterial total RNA samples. We also expanded the probe design to remove adult, fetal and embryonic hemoglobin transcripts from multiple derivate blood samples for which globin can constitute up to 70% of total mRNA transcripts.
Using strand-specific RNA sequencing we measured depletion efficiency, library complexity, transcript coverage and transcript expression before and after depletion across a wide range of samples and RNA input amounts. We achieved high depletion efficiency (up to 99.9 % rRNA depletion) with minimal off target effects. We detect high number of transcripts, with even coverage across the transcript length and retention of transcript complexity even at the lowest inputs. The method works efficiently in low input and highly degraded total RNA including that from formalin-fixed-paraffin-embedded (FFPE) samples.
We conclude that the reduction of abundant transcripts for RNA-Seq studies significantly increases the ability to detect true biological variations that could not be detected in non-depleted samples. The method described here is a reliable and simple solution that greatly improves sensitivity in transcriptome RNA-Seq studies. Furthermore, it is amenable to high throughput sample preparation and robotic automation for easy implementation in a clinical setting.
(138) Impact of fecal microbiome extraction technique on relative abundance of genera within expected and unexpected communities
Microbiome analysis has gained significant interest as sequencing technology has improved, allowing for the examination of microbial communities often believed to play a key role in human health and disease. 16S rRNA sequencing analysis, utilizing various publicly available bioinformatic tools, allows the study of diversity in microbial communities. It has previously been shown that extraction techniques are impactful for the analysis, however, these studies have often been performed with a single cultured sample, or with evenly distributed mock communities, failing to provide variability often seen with human source samples and excluding the impact of amplification and/or sequencing related bias.
Within this study, we analyzed six different extraction protocols, utilizing commercially available reagents, with a naturally collected fecal sample, two mock communities, all of uneven species distribution, and a manufactured extraction control, of known species distribution. In addition, six sequencing controls, already extracted DNA of variable concentration and percentage of species distributed within each community, were utilized to determine the additional bias introduced by amplification and/or sequencing reagents.
Significant differences were noted in alpha and beta diversity of samples extracted using each protocol, particularly as compared to the expected values, consistent with previous findings. Additionally, there were unexpected genera found within these communities indicating contamination uniquely introduced by each protocol. Trends were noted among different kits, in either over- or under-representation of specific genera of communities. Similar trends were observed in the results of sequencing controls, indicating the contamination and/or bias introduced after extraction. Overall, these findings illustrate the importance of utilizing controls at multiple stages of the process, to better assess the impact of the findings of any human samples.
(140) Linking the Resistome to the Microbiome: A Culture-Free Method Links Plasmid, Virus, and Antimicrobial Resistance Genes to their Hosts in Complex Microbial Populations
Background: The rapid spread of antibiotic resistance is a global health threat. A range of environments have been identified as reservoirs of the antibiotic resistance genes (ARGs) found in pathogens, but we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to an inability to identify the bacterial hosts of ARGs and the mobile genetic elements that mediate horizontal gene transfer due to the loss of intra-cellular contiguity upon DNA extraction.
Methods: In two recent studies we describe the application of proximity-ligation methods for the determination of the in situ host range of numerous ARGs, viruses, plasmids, and integrons within complex microbiome samples. This method forms physical junctions between sequences present within the same cell prior to DNA extraction. Subsequent sequencing generates a dataset that robustly connects mobile elements to their hosts and can assemble de novo genomes from mixed communities.
Results and Conclusions: Our application of this technology to complex wastewater and rumen samples yielded hundreds of novel ARG-, virus-, and plasmid-host interactions, as well as over a thousand new microbial genomes. These studies highlight the power of the proximity-ligation approach to deconvolving microbiome samples and foreshadow the development of rapid culture-free strategies for tracking and managing the spread of antimicrobial resistance.
Linking the Resistome and Plasmidome to the Microbiome; Thibault Stalder et al., Dec. 2018, bioRxiv
Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation; Derek Bickhart et. al., Dec. 2018, bioRxiv
(308) High throughput profiling of glycans released from therapeutic glycoproteins via micro-permethylation.
Glycans significantly influence the therapeutic efficacy of glycoprotein therapeutics by regulating its stability, serum half-life, immunogenicity and importantly, biological activity. The demand for the qualitative and quantitative characterization and validation of glycosylation on therapeutic glycoproteins which includes antibodies, vaccines, biomarkers etc. are increasing rapidly. One of the classical yet most informative derivatization process for the glycan characterization is the permethylation of glycans released from the biomolecules and subsequent characterization by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Electrospray Ionization (ESI-MS). Even though the manual process of permethylation and subsequent processing of glycans are still highly prevalent, some recent attempts were made in the automation of the permethylation process. For a cost-effective, simple, sensitive and rapid permethylation kit for the permethylation of glycans released from the glycoproteins, we developed a micro-permethylation procedure. Here, we report a protocol for the automation of permethylation reaction in microscale that allows processing of samples in a minute amount in a high throughput fashion with minimal handling skills. The protocol involves releasing of N/O-glycans from glycoprotein samples, the permethylation reaction in microscale, extraction of permethylated glycans directly in a 96 well plate or microcentrifuge tube within significantly short processing time and efforts. The permethylated glycans are subsequently analyzed by mass spectrometry. The workflow was applied for the permethylation and subsequent in-depth characterization of N/O-glycans released from both standard glycoproteins such as Fetuin and Ƙ-casein, and also therapeutically relevant human serum IgG and transferrin. Also, the procedure involves the use of less hazardous chemical reagents and generates less amount of harmful waste materials in comparison to other similar methods. The protocol can easily be automated for the high throughput screening of glycosylation on a large sample set of glycoprotein drug profiling and clinical glycan biomarker studies with convenient, faster, and reliable characterization.
(162) Supporting Single Cell RNA-seq Analysis at Harvard - A Community Approach
Recent advances in single cell transcriptomics make it possible to examine the gene expression profiles of thousands of individual cells, providing unprecedented insights into tissue heterogeneity, development and pathogenesis. In 2015, the Harvard Chan Bioinformatics Core (http://bioinformatics.sph.harvard.edu) teamed up with the Harvard Medical School (HMS) Single Cell Core (https://iccb.med.harvard.edu/single-cell-core) to standardize data analysis for the InDrop droplet barcoding system and prepare for projected demand within the Harvard community. Here we describe our approach to building single cell analytical expertise and infrastructure through our partnership with the Single Cell Core and multiple research labs. We outline the challenges we faced and our current best practices for data analysis (quality assessment, quantitation, clustering, visualization, and differential expression). Our pipeline, implemented within the bcbio-nextgen framework (https://bcbio-nextgen.readthedocs.io/), handles multiple UMI schemes to accommodate different single cell technologies (e.g. Drop-seq, Seq-well, Bio-Rad ddSeq, etc.). We also describe our approach to managing single cell projects, with their longer analysis times, increased complexity and need for rigorous experimental design, data management, computing infrastructure and methods evaluation. All of these require close collaboration and frequent communication with the bench biologists generating the data. Due to these factors, we have expanded our bioinformatics training program to include modules on single cell RNA-seq. With this program, we hope to develop analysis expertise within the community and an understanding of the methods and intricacies inherent in the technology - ultimately leading to better designed and more successful single cell RNA-seq experiments.
(156) RipTide™ Ultra High-Throughput Rapid DNA Library Preparation for Next Generation Sequencing
Whole Genome Shotgun Sequencing has become the tool of choice for microbial genome analysis. Rapidly declining costs of sequencing, data analysis, data storage and database access will continue to drive adoption. Library construction has not kept pace with these advancements, with costs of preparing a next generation sequencing (NGS) library often exceeding the cost of sequencing. Popular methods of library construction for NGS include fragmentation, end-repair and adapter ligation, and transposase-mediated adapter insertion. The Riptide High Throughput Rapid DNA Library Prep is distinctly different in its approach because it relies on polymerase-mediated primer extension for library preparation. The initial step of the prep, involving primer extension with barcoded random primers, is performed in a 96-well plate. Each well of the plate contains primers with a unique barcode; consequently, the library generated from each well is uniquely identifiable and can be bioinformatically traced back to the original sample after sequencing. Following this step, the primer extension products are combined into one pool and all subsequent steps, including second strand synthesis and PCR, are performed with the single pool. The library prep is fast, easily automatable and can be tuned to genomes of high and low GC content. With automation, 960 samples can be processed in a single day. The technology will aid genetic research by helping to increase sample throughput and by reducing processing steps and operating costs. Presented here is RipTide High Throughput Rapid DNA Library Prep sequencing data generated from multiple microbial genomes.
(102) Detecting Genomic DNA Contamination in RNA Samples Using the Bio-Rad Droplet Digital PCR System
Droplet Digital PCR (ddPCR) allows ultrasensitive and accurate absolute quantification of nucleic acid target sequences through the partitioning of the conventional reaction volume of standard PCR into 20,000 discrete, uniform “mini-reactions”. This massive reaction partitioning increases signal-to-noise by reducing the competition from high copy templates. Standard PCR reactions are prepared and then subjected to partitioning via emulsification in water-oil based droplets using microfluidics. Each droplet represents a single PCR reaction and following the amplification reaction of the nucleic acid target, the fluorescent signal generated within each droplet is detected and quantified. Sample partitioning facilitates the specific detection of single template molecules and eliminates target template competition, reducing reaction inhibition and increasing sensitivity. The BioRad QX200 Droplet Digital PCR system provides absolute quantification of target DNA or RNA molecules in complex sample backgrounds. To test the ability of this instrument to detect very low amounts of contaminating genomic DNA (gDNA) in a complex RNA sample ddPCR primers were designed to specifically amplify only the intron region of the human actin gene. Contaminating gDNA in RNA preps can cause inefficient polyA selection prior to cDNA synthesis and lead to RNASeq artifacts. Varying amounts of human gDNA was spiked into human RNA in duplicate reactions and the level of human actin intron DNA region in the mixed samples was analyzed using BioRad QX200 instrument. Spiked gDNA contamination as measured by the presence of the actin amplicon was reproducibly detected with a threshold of 0.5%. The appropriate sized single intronic amplicon was detected in gDNA alone, while no amplicon was detected in the RNA sample alone. These results indicate that ddPCR can be used to assess gDNA contamination in total RNA preparations. We will discuss the use of this ddPCR method for assessing gDNA contamination in customer RNA samples submitted to the ICBR GE for preparation of RNASeq libraries.
(210) cellPhoresis: A dielectrophoresis based technique for rapid, nondestructive cell separation and analysis.
Analysis and identification of live cells is necessary in many fields; water quality, cell therapy, drug development, clinical diagnostics, biofuels, and brewing are a few examples. Existing methods alter or destroy the cells during analysis, take too long (2-3 days), or require labeling and/or specific markers. We present a microfluidics and electrokinetics based technique, cellPhoresis, that does not destroy the cells, does not require labeling or specific markers, and takes under 30 min to go from sample loading to results. Cells are captured and concentrated at discrete locations along a microfluidic channel, and quantified by light microscopy. Other quantification methods such as impedance or Raman spectroscopy can also be implemented. Subtle phenotype differences were identified in bacteria and mammalian cells in various applications.
(400) Adoption of Evaluation and Assessment Data Management Tools for a Growing Core Facility with Program Grants
The Biomolecular Research Center (BRC) at Boise State University is a core facility focused on the study of biomolecules with emphasis on proteins and their molecular interactions. The BRC is also the administrative center for multiple programmatic and individual researcher-initiated biomedical research grant awards including the NIH Center of Biomedical Research Excellence (COBRE) in Matrix Biology Program, Idaho INBRE Program, Mountain West CTR-IN Program, and awards from other agencies including AHA, NSF, and the M.J. Murdock Charitable Trust. These awards have made a significant impact on research and research infrastructure growth at Boise State. The Idaho INBRE program acted as the catalyst for the creation of the BRC through the establishment of infrastructure, funding, leadership, and mentorship. As a result, Boise State received its first COBRE grant in 2014. The growth in biomedical research and shared core facility use required new approaches to information management for reporting, assessment, and evaluation. Data management goals include: 1) Collection and management of annual reporting information from investigators, staff, and students; 2) Implementation of software for analyzing synergy between our program’s management strategy and investigator success; and 3) Consolidation of core facility’s management, billing, and reporting capabilities into one cohesive system. This approach has streamlined administrative functions and increased efficiency of information management and our ability to assess and evaluate our center’s impact on investigators’ success as well as programmatic success. Finally, adoption of these best-practices has lessened administrative burden, more effectively managing day-to-day center operations while increasing transparency of core facility activity. The substantial burden of data management and assessment requirements for research centers and programs necessitates early consideration of a thoughtful and meaningful strategy for data management. Flexibility must be maintained to allow continuous improvement to approaches used for assessment and evaluation in ways that streamline dissemination of outcomes to all stakeholders.
(104) End-to-end next-generation sequencing for strain sub-typing and epidemiological analysis: an exploration of relatedness and virulence in Streptococcus pyogenes and SDSE
Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging human pathogen causing life-threatening invasive infections including the streptococcal toxic shock syndrome. Laboratory reporting of SDSE is poorly understood and little insight regarding clonal relationships of SDSE exist. We hypothesize that virulence genes between Streptococcus pyogenes (GAS) and SDSE exist; contributing to an evolving disease spectrum of SDSE infections. Using whole genome sequencing of 42 SDSE isolates from clinical infections and asymptomatic volunteers we describe the genetic relatedness and overlap between GAS and SDSE and described virulence factors associated with GAS isolates within SDSE.
We demonstrate here how the use of whole-genome sequencing and genomic analysis helps to evaluate trends in disease and disease sub-populations of SDSE.
NGS and data analysis was provided by the commercial GLP- and CLIA-certified service provider CosmosID and included i) library preparation and genome sequencing ii) unambiguous strain-level taxonomic classification via CosmosID’s industry-leading databases and bioinformatics platform, iii) genotyping, iv) the comprehensive characterization of antimicrobial resistance, plasmids and virulence, and v) a detailed analysis of relatedness between isolates and previously reported genomes in order to better delineate transmission events.
We present the range of Group A Streptococcus (GAS) virulence genes that were found in each of the 3 study groups. Interestingly, the groups (pharyngeal, extra-pharyngeal, and control) were not found to be distinct based on phylogenetic analysis alone. Therefore further comparisons of virulence factors found among the isolates were made, particularly exploring the importance of GAS virulence factors that can be found in SDSE and possibly by evaluating trends in invasive disease produced by SDSE over time.
(310) Incorporating In-Source Fragments Improves Metabolite Identification Accuracy in Untargeted LC−MS and LC−MS/MS Datasets.
In untargeted metabolomics experiments library search engines detect metabolites using several features, including precursor mass, isotopic distribution, retention time, and MS2 fragmentation. Matching acquired MS2 to library spectra is vital as numerous compounds share molecular formulas, resulting in identical precursor measurements and similar retention times. However, many metabolomics experiments are still collected using LC-MS only, and even in LC-MS/MS experiments many precursors lack MS2 spectra due to the stochastic nature of data dependent acquisition. We observe that when metabolites ionize they can produce unanticipated MS1 features resulting from neutral losses, in-source fragmentation, multimerization, and adducts. Here we present a new approach to leverage these measurements to identify metabolites when MS2 spectra are of low quality or not available. We processing datasets of 75 known standards mixed with whole yeast lysates to strip them of their MS2 scans to produce a “gold-standard” MS1-only data set of a complex metabolome with known targets. For each dataset we determined the proportion unambiguous annotations (where the correct annotation had a higher score than other potential annotations) and unmistakable annotations (where the correct annotation was the only valid annotation detected). We found that incorporating in-source fragments improved these metrics for both MS1-only (increasing from 60% to 73% unambiguous and 40% to 65% unmistakable matches) and MS2 datasets (from 79% to 84% unambiguous and 41% to 60% unmistakable). Unexpectedly, in these data we observed that the MS2 spectra were less useful than in-source fragment data for improving identification accuracy. We believe this is largely because the low-resolution iontrap MS2 spectra collected in this experiment show significant noise, which diminishes spectral match scores and allows other candidates to outscore the correct identifications. We suspect that noise is less likely to affect MS1 peak groups because they are generated from data aggregated across multiple high-resolution MS1 scans.
(168) NEXT-GEN SEQUENCING OLIGOS (NGSO) FOR CONSISTENT AND EFFICIENT SEQUENCE ASSEMBLY
Target identification is often the first step in any drug discovery workflow. An efficient and unbiased screen of the entire genome for specific cellular phenotypes can lead to rapid identification of potential molecular targets for therapeutic intervention. Whole-genome screening applications often require nextgeneration sequencing (NGS) of cell populations with the desired phenotype. Regardless of NGS platform, universal and index adapter sequences are required for the proper assembly of sample fragments. Adapters – especially index adapters since they contain the multiplex identifier (MID) or barcode – containing too high a proportion of truncated sequences (unacceptably low purity) or too high a proportion of other adapter sequences (excessive cross contamination) can lead to compromised sequence read integrity as well as excessive adapter dimerization and improper sequence assembly, respectively, during multiplexing experiments. These types of problems are undetectable until the data analysis stage, which makes them costly in terms of time and money. Therefore, the production process used for the adapter sequences is critical for a successful sequencing run. Affordable, custom Next-Gen Sequencing Oligos (NGSO) are manufactured under rigorous conditions to ensure suitable purity and low cross contamination and to meet research, commercial, and molecular diagnostic needs. Our approach eliminates failed sequencing runs that could have resulted from poor adapter quality. Customer testing has shown our NGSO to be 70% less expensive than a popular adapter kit (on a per library preparation basis) as well as capable of preparing libraries with adapter dimers and cross contamination as low as 6.28% and 0.015%, respectively. Use of NGSO will be of tremendous benefit to drug discovery workflows that require next-generation sequencing.
(204) QUALITY CONTROL OF MODERN FLUORESCENCE MICROSCOPES
Although quality control (QC) of fluorescence microscopes is a topic that appeared more than fifteen years ago in academic laboratories  and national regulatory agencies , it is still topical as it was for example in the program of the Core Facility Satellite Meeting of the 15th and 17th international ELMI meetings in 2015 and 2017. Due to the increasing complexity of the instrumentation used for confocal and wide-field fluorescence microscopy, national metrology institutes , microscopes manufacturers , and core facilities  have gotten involved in identifying, making and/or testing different tools, both hardware and software, to assess the numerous aspects of fluorescence microscopes. Indeed, QC is important: (i) for core facilities, to assure the performances of the microscopes they make available to the end users; (ii) for microscope manufacturers, to guarantee the microscopes’ specifications and to improve maintenance; (iii) for end users to remove the bias introduced by the microscopes in their experiments and make their experiments reproducible.
We have developed a new process that enables the etching of long-term stable fluorescent patterns with sub-micrometer sizes in 2D and 3D inside glass. Based on this new process, fluorescent patterns and dedicated image analysis algorithms are shown to be suitable for complete and quick QC of fluorescence microscopes . Non-exhaustively, this new solution enables the QC of: illumination power, illumination inhomogeneity, field distortion, chromatic shifts, resolving powers...
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(134) High-throughput and automated procedures for microbiome analysis of stool and soil
Microbiome studies often require processing hundreds or thousands of samples in order to identify large-scale differences in the community composition of the target sample type. Large scale studies of this type are enabled by the advent of highly multiplexed 16S next-generation sequencing, for which efficient sample extraction techniques are needed. Two of the most common sample types studied are soil (environmental studies) and stool (human microbiome studies). These studies of the environmental and human microbiomes are challenged by the fact that soil and stool are extremely difficult sample types due to their high abundance of inhibitory substances, which are co-purified and have the potential to inhibit enzymatic reactions.
The second generation of the QIAGEN/MO BIO Inhibitor Removal Technology in the PowerSoil Pro and PowerFecal Pro kits addresses these challenges and efficiently removes PCR inhibitors from difficult soil and stool samples. These kits have been adapted to high-throughput processing methods, including 96-well plate-based as well as magnetic bead-based protocols which allow automation on a wide variety of platforms. High-throughput bead beating homogenization allows rapid lysis by a combination of mechanical and chemical methods. Inhibitors are removed by a specialized precipitation step, after which DNA purification can be carried out in 96-well plate formats. This technology enables high-throughput processing of stool and soil samples for downstream applications such as PCR amplification and 16S rDNA sequencing.
These procedures provide equivalent yields and purity to single spin column workflows, while enabling much higher throughput in equivalent times. 16S sequencing shows equivalent community composition to manual methods, as shown by OTU analysis as well as alpha and beta diversity measures. These new procedure are ideal for high-throughput sample processing of both environmental and stool samples.
(302) A Rapid and Simple Proteomics Digestion Protocol
Many proteomics sample preparation workflows are cumbersome involving numerous steps including denaturation, reduction, alkylation, and overnight protease digestion. Lengthy steps require 3-4 hours of hands-on lab work followed by 16-20 hours of incubation. Minor deviations or environmental fluctuations potentially introduce variability to sample preparation, which in turn could impact the reliability and reproducibility of the mass spectrometry results. We sought to develop a rapid trypsin digestion workflow that would produce reliable and reproducible results.
Individual proteins and complex protein mixtures were digested with solution-stable trypsin in microtubes using an optimized digestion buffer for one hour at 60°C, both with and without reduction and alkylation. Cell lysate samples with SDS were digested with a filter-assisted sample preparation (FASP) method using the optimized buffer at 60°C for one hour. For comparison, all samples were also digested overnight using traditional in-solution or FASP methods as controls. Digests were analyzed using a nanoLC system coupled to a Thermo Fusion Tribrid system. Data analysis was performed using Mascot and Scaffold software.
The rapid digestion of individual protein samples yielded comparable sequence coverage results to those obtained by the samples that were digested overnight. It is noted that proteins with high cysteine content may still require reduction and alkylation for efficient digestion. Similarly, complex samples digested according to the optimized one-hour digestion method (without the need for reduction, and alkylation) produced comparable protein/peptide identifications as obtained for the complex control samples.
We present an optimized digestion buffer and a rapid workflow consisting of trypsin digestion at elevated temperature, which prepared reliable and reproducible samples in one hour for a wide variety of sample substrates.
(324) Spatial Mapping of Lipids and Neurotransmitters in Rat Brain Section Using DESI Ion Mobility Mass Spectrometry
Spatial mapping of small molecules, such as neurotransmitters, alongside lipids, can increase our understanding of biological functions of those molecules within the brain. Desorption Electrospray Ionization (DESI) is an ambient ionization technique that can spatially profile the distribution of molecules in research tissue samples. Here we present the utility of DESI imaging to simultaneously detect lipids and neurotransmitters directly in brain tissue samples.
Rat brain was harvested and flash-frozen in liquid nitrogen before cryosectioning. Coronal tissue sections (8 microns thick) were mounted on regular glass microscope slides, vacuum dried, and analyzed without any further sample preparation. The DESI imaging platform coupled with a high definition mass spectrometer (HDMS) with ion mobility separation was employed to obtain ion intensities of small molecules and lipids over the entire tissue. DESI Imaging data were collected and processed on a high definition mass spectrometer with ion mobility separation. DESI acquisitions were performed using methanol and water as a DESI spray solvent.
The ambient nature of DESI allowed for MS imaging without any matrix application or extensive sample preparation steps. Molecular maps were processed and overlaid with an optical image of the tissue to co-register the molecular distribution based on the anatomical features of the brain, such as the corpus callosum. Small molecules such as amino acids and neurotransmitters were simultaneously detected along with lipids. Molecular identification was aided using high mass accuracy database searches against LipidMaps and HMDB. In addition to the accurate mass and high-fidelity isotopic distribution, collisional cross sections (CCS) or drift time data obtained during ion mobility separation was used to improve confidence in detected molecules. Ion mobility measurements before the MS measurment increased the coverage and added selectivity which helped identification This preliminary work indicated the utility of DESI imaging for clearly distinguishing localized metabolites and lipids to provide insights for neuromolecular research.
(304) Characterization and cytotoxic activity of Non-Specific Lipid Transfer Protein (NsLTP) from Fennel (Foeniculum vulgare) seeds
Fennel (Foeniculum Vulgare), is a flowering medicinal plant that belongs to the family Umbelliferae (Apiaceae). It is native to southern Europe and Mediterranean region with long history of use by humans as a spice, medicine, and fresh vegetable. Despite its popularity for its medicinal value, there are limited scientific studies about the structure and functions of proteins isolated from the seeds and their pharmacological activities. Yet majority of data on literature is based on the crude extracts and/or essential oils. Thus, this project explores the complete structure of Non-Specific Lipid transfer protein (NsLTP) isolated from fennel seeds along with their cytotoxic activities. The protein was extracted using Tris/HCl pH 8 buffer and purified by the combination gel filtration and reverse phase HPLC. The purity was confirmed by SDS-PAGE gel electrophoresis and intact mass analysis by MALDI-TOF mass spectrometry. The purified NsLTP-protein was modified by 4-vinyl pyridine followed by digestion with trypsin. The tryptic digest was then separated by reversed phase HPLC. The primary structure of NsLTP was established by combining N-terminal amino acid sequence of intact chain and tryptic peptides by Edman degradation in automated protein sequencing system. The data suggest that Fennel NsLTP is a monomeric protein of 10kDa based on SDS-PAGE electrophoresis. Multiple sequence alignment performed using Clustal Omega depicted a sequence similarity with Daucus carota and Actinidia chinensis. The MTT assay result revealed that Fennel NsLTP led to dose dependent cytotoxic effect in MCF-7 cells with an IC50 value of 98.1µg/ml. Besides, mRNA expression of the genes Bax, EGFR, VEGF, Caspase-3, Survivin, MMP, and Bcl-2 were modulated as assessed by real time PCR.
(152) QIAseq multimodal analytical technology: Single workflow DNA/RNA NGS library prep and analysis
Integrated genomic and transcriptomic analysis provides comprehensive insights in cancer research. Thus far, integrative only occur at the insight stage, with the library construction, sequencing and data acquisition occurring in separate, sequential experiments. To streamline this process, we have developed a novel NGS workflow, which allows fully integrated targeted library construction and analysis of multi-omics biomarkers (DNA SNV, RNA SNV, RNA fusion and RNA expression) from total nucleic acid samples.
QIAseq multimodal workflow starts with total nucleic acid (NA) extraction from various sample types including FFPE curls, whole blood, cells and fresh biopsies. RNA in the sample is reverse transcribed and all subsequent library construction steps are conducted to prepare genomic DNA and RNA libraries, with each NA type being uniquely tagged. Separate DNA and RNA amplification steps are performed to facilitate optimal concentration adjustments for the combined RNA and DNA libraries for sequencing. The core technology leverages single primer extension (SPE) chemistry for efficient targeted enrichment and unique molecular indices for enhanced error correction in variant calling and more accurate quantitative expression analysis.
To demonstrate the capabilities of this technology, we designed a Hema-oncology panel with 465 primers for selected genes, hotspot exons and relevant gene fusions. We demonstrate our multimodal workflow using total nucleic acid mixtures from both cell lines and FFPE samples. SNV/SNP, fusion and expression analysis can be reliably analyzed from both DNA and RNA simultaneously using this integrated workflow. Taken together, multimodal technology realizes simultaneous library preparation and analysis from DNA and RNA, which reduces sample consumption, streamlines the NGS workflow and allows confident variants calling and precise gene expression analysis.
(106) High-Throughput, High MW DNA Extraction and Size Selection for Long-Read Sequencing
Long-read sequencing technologies from PacBio and Oxford Nanopore are rapidly becoming the de facto methods for de novo assembly, phasing, and structural variant analysis. These capabilities are predicated on high purity, high molecular weight (HMW) DNA input. However, the gold-standard plug lysis method is tedious and exceedingly slow while conventional spin columns and magnetic beads shear DNA resulting in compromised read lengths.
We present novel methods for rapid HMW DNA extraction and library preparation based on our Nanobind magnetic disks. Each disk (1-5 millimeter diameter) is covered with a high density of micro- and nanostructured silica that protects DNA from damage and enables high extraction yield and purity. DNA extraction is performed using a simple bind, wash, and elute process that can obtain HMW (300+ kb) and megabase (1+ Mb) DNA in less than 1 hour for most sample types. Automated HMW DNA extractions can be performed using ThermoFisher KingFisher instruments.
HMW DNA extraction and sequencing results from a wide variety of sample types including cultured cells, gram-negative and gram-positive bacteria, whole blood, plants, insects, and tissues are shown. PacBio SMRT sequencing on Sequel System typically results in subread length N50s up to 40 kb and throughput up to 15 Gb per flow cell. Sequencing on Oxford Nanopore MinION/GridION typically results in read length N50s of 50 kb with yields up to 10 Gb per flow cell. Megabase length reads are demonstrated using the Oxford Nanopore Rapid Sequencing Kit.
In addition to DNA extraction, Nanobind can be used for tunable size selection up to 10 kb with >80% recovery of high MW DNA and >99% removal of short DNA. Gentle purification and high recovery ensure a greater number of long-reads >50 kb. This method enables library prep to be completed in less than 4 hours.
(170) Automated Unbiased Metagenomic DNA Extraction for Long-Read Sequencing
Long-read DNA sequencing is being touted as the “next next-gen” sequencing due to its affordability, ease of use, and increased output of more accurate data compared to traditional next-generation sequencing technologies. For example, the advantages of Nanopore long-read sequencing include the capacity to generate very long reads with remarkable speed and portability, spanning tandem-repeat regions, which resolves ambiguity during genome assembly. However, extracting inhibitor-free high molecular weight (HMW) DNA suitable for long-read sequencing has always been a challenge due to DNA fragmentation during extraction caused by physical and enzymatic (DNases) breakage. Here we present an automated HMW DNA extraction pipeline that combines magnetic bead-based DNA extraction with the Microlab STAR™ liquid handler. Magnetic bead chemistry allows automated processing by retaining HMW DNA during stringent wash steps, leading to high quantities of long-read sequencing-ready DNA. To test the efficacy of this automated workflow, HMW DNA was isolated from a well-defined mixture of bacteria and yeast cells and subsequently sequenced using the MinION™ platform. Using this method, we achieved 1M reads, 8 Gb throughput, with average read-lengths of 8 kb, and over 100 kb recorded. Moreover, we find that the extracted microbial profile and proportional composition closely matches the theoretical composition. Overall, we have developed an automated method for HMW DNA extraction that shows unbiased microbial lysis that is compatible with long-read nanopore sequencing.
(202) K-means Spectral Unmixing for Multi-channel Imaging and Image Analysis Platform at a Core Facility
There is an increasing need of multi-channel fluorescent imaging to simultaneously visualize different biological structures and the dynamic interactions between them. However, due to the overlapping emission spectra of many commonly used fluorophores, many times fluorescent images have bleed-through problems when a fluorophore in one channel crosstalk with another channel, leading to a false positive detection. This problem is almost unavoidable when the samples are stained with three or more dyes, and especially imaged under multiphoton microscope in which case the fluorophores two-photon excitation spectra are usually broader than one-photon. A lot of methods have been used to do spectral unmixing, such as non-negative matrix factorization and similarity unmixing. However, they either need known emission spectra or assume one fluorophore per pixel.
To help users solve channel bleed-through problem, the Multiphoton Imaging (MP) core at URMC developed a novel method by using K-means clustering to separate mixed channels and clear up images. With a given multiphoton image, pixels are classified into K different clusters based on their intensities in all the detection channels, with a minimal within-cluster sum of squares. Pixels bleed into another channel will be separated out from that channel’s cluster due to a different “intensity signature”. Surprisingly, this method also works very well to remove background noise and autofluorescence by treating the background or autofluorescence signal as an additional cluster. Furthermore, as a core, we integrated this tool into Imaris for users without any programming knowledge to use it easily.
This newly developed tool for image pre-processing has been used by many users at the MP Core and is going to be applied for more image modalities such as confocal. The success of this development has initiated our image analysis service, and we are continuing building an Image Analysis Platform with the focus of more customized image analysis solutions.
(132) Generating Standards for Epigenetics Research: The FDA’s Working Group on Epigenome Quality Control (EpiQC)
Detection of DNA modifications such as 5-methylcytosine (5mC), N6-methyladenosine (m6A), and 5-hydroxy-methylcytosine (5hmC) are essential for understanding the epigenetic changes that guide development, cellular lineage specification, and disease across all kingdoms of life. However, a proliferation of molecular methods to interrogate these modifications and their phased haplotypes (epialleles) have created the need for standardized materials, methods, and rigorous benchmarking. These can then further enable and improve their applications to clinical and research projects. Here we report a multi-platform, multi-site assessment and global resource for epigenetics research from the FDA’s Epigenomics Quality Control (EpiQC) Group. The general study design primarily leverages 7 human cell lines, publicly available from the National Institute of Standards and Technology (NIST) Genome in a Bottle (GIAB) Consortium. Our primary focus was on cytosine modifications found in mammalian genomes (5mC, 5hmC). Each sample was processed for whole-genome bisulfite sequencing (WGBS), targeted methylation sequencing, oxidative bisulfite sequencing (oxBS), and an Enzymatic Methyl-seq (EM-seq) deaminated reference data set, followed by a rigorous assessment and comparison to the 450K, 850K Illumina methylation chips. An increasingly popular chromatin conformation assay, ATAC-Seq, was also studied. Our goals are to identify best-practices, data types, laboratory methods, and computational algorithms to inform ongoing efforts in epigenomics research, DNA modification detection algorithm development. We hope these results simplify applications of 5C modification information in the contexts of gene regulation, clinical diagnostics, prognostics and systems biology.
(118) Bead-Linked Transposome Technology: Ushers a Next Era in library prep enabling a normalization-free workflow for whole genome and flexible targeted resequencing
We describe a bead-linked transposome technology used for preparing libraries for WGS (Nextera DNA Flex) as well as targeted enrichment (Nextera Flex for Enrichment). This methodology utilizes a known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA. This therefore offers broad applicability, supporting a wide spectrum of DNA input ranges as well as integrated extraction of blood and saliva samples generating normalized libraries for sequencing to facilitate a quantification-free workflow. Libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. On-bead tagmentation chemistry is also applicable for targeted sequencing and supports a wide range of DNA input amounts, various sample types, and a broad range of applications, including fixed panels, custom panels, and whole-exome sequencing. Furthermore, Nextera Flex for Enrichment is compatible with Illumina and third-party enrichment probes/panels, which enables content portability. The innovative Nextera Flex for Enrichment solution combined with the power of Illumina SBS chemistry provides an optimal targeted enrichment and exome sequencing experience. The bead-linked transposomes can therefore be effectively used for WGS as well as for targeted resequencing.
(172) Next Generation Whole-Genome Amplification Methods
Preparation of amplified genomic material from small amounts of DNA or single cells is extremely important in assisting research involving genetic analyses of clinical samples aimed at identifying the best treatment regimen and molecular diagnoses of diseases such as cancer. Technologies that allow for accurate and reproducible detection of single nucleotide variations (SNVs) and copy number variations (CNVs) in genomic material from limited samples need to do so with high fidelity and with high genome coverage. Additionally, they should be flexible enough to be used in a variety of analysis platforms. To address these needs, we have developed the SMARTer® PicoPLEX® WGA V2 System (PicoPLEX WGAv2), a platform-agnostic whole-genome amplification system, and the SMARTer PicoPLEX Gold Single Cell DNA-Seq Kit (PicoPLEX Gold), a complete cells-to-library solution for Illumina® sequencers. These systems use optimized enzymes, primers, and protocols for optimal sequencing coverage, uniformity, and accuracy to detect SNVs, all while increasing the resolution for CNV detection relative to previous versions. Both systems maintain the technology’s simple workflow and unmatched cell-to-cell reproducibility that is a hallmark of the PicoPLEX technology. In this study, we have demonstrated CNV detection to 5.5-MB mb resolution at a depth of 1 million read pairs in a single cell, with validated copy number gains and losses. SNV detection and reproducibility are shown to be superior to competitive technologies.