Concurrent Scientific Session (Genomics): MGRG (Metagenomics) and ABRF-NGS (The NGS Study Consortium)
A Multi-Platform Assessment of DNA Sequencing Reproducibility using Human and Bacterial Genomes
The ABRF Next-Generation Sequencing (NGS) Study examines well-characterized human and bacterial genomic reference material to evaluate the reproducibility of sequence data generated using industry standard DNA sequencing platforms. This study is not a “bake-off” between instruments, but rather an effort to establish a reference dataset that will allow users of high-throughput platforms to evaluate the performance of their instruments as protocols and technologies rapidly evolve. In collaboration with the Genome in a Bottle Consortium and other sequencing community stakeholders, the ABRF-NGS group has sequenced NIST DNA Reference Materials derived from cell lines of Personal Genome Project (PGP) contributors, as well as ATCC bacterial genomic microbiome standards, using a range of technologies including Illumina and Thermo Fischer. For each instrument, the study design included inter- and intra-lab replicates to facilitate different levels of output comparison. All data were processed through an alignment and variant analysis pipeline using Sentieon, and examined for sequencing accuracy, quality divergences, and performance within genomic contexts such as GC extremes, areas of low complexity, and "difficult" regions of the genome that include the “Classes of Evil”. Different platforms exhibited relative strengths and weaknesses relative to one another and with respect to different samples. These comparisons, which are available in a dynamic, publicly accessible web resource, can be used as a baseline for researchers to evaluate their own NGS outputs.
The MOSAIC Standards Challenge: Capturing the extent and sources of variability in NGS-based microbiome profiling protocols and analyses
Several studies have documented the significant impact of the multitude of methodological variables in microbiome research and the effects on variability and overall consequences of NGS-based microbiome data. In most cases, these variables impede reproducibility of results and the ability of researchers to perform comparisons between independent studies that use differing techniques and data pipelines.
To tackle this issue, the MOSAIC Standards Challenge will provide and ship 700 sets of fecal aliquots and DNA reference material, at no cost to Challenge participants, to any lab in the world. Each lab will perform their preferred sequencing protocols and upload their sequencing and metadata data to Mosaic. Data will allow to comparisons of the different protocols and analysis methods for taxonomic profiles.
Metagenomics Research Group Study: Characterizing The Whole Cell Microbial Reference Standard using RNA and DNA-Sequencing
As microbiome and metagenomic studies span nearly all aspects of science, the need to characterize the limitations and strengths of the full laboratory process is critical. This includes DNA and RNA extraction efficiencies, type of sequencing, and bioinformatics, to name a few. Presently, a commercial accurately enumerated whole cell microbial standard does not exist, except for the MGRG WCM Standard that is near completion. The minimum design characteristics included 1) accuracy cellular enumeration of each species, 2) intact cellular integrity and morphology that came be confirmed by microscopy and staining, 3) preserved, 4) fully sequenced genomes, 5) various Gram reaction and GC content, and 6) able to maintain cell wall digestion with lytic enzymes. In this study, we have performed initial experiments to characterize this standard using various kits and sequencing approaches. We will present both RNA and DNA extractions and sequence results as well as open up the discussion to the audience.