Concurrent Scientific Session (Genomics): Epitranscriptomics

Abstracts

Signal analysis of nanopore RNA sequencing to interrogate poly(A) tails and post-transcriptional modifications

Speaker: Roham Razaghi
Track:

Although development of high throughput RNA sequencing technologies has allowed significant advances in our understanding of transcriptome complexity and regulation, certain questions remain intractable with conventional short read cDNA sequencing. Native RNA sequencing technology can address these difficult questions; including identity and frequency of splice variants, full-length poly(A) tail assessment, base modifications and transcript haplotyping. To accomplish this, we leveraged our extensive consortia-generated GM12878 native RNA dataset to showcase this exciting technology and the promise it holds for the direct and simultaneous interrogation of these RNA features.

 

We have applied tools to directly interrogate the raw electrical current data from nanopore sequencing to examine these questions.  Specifically, we have begun to empirically establish how different modifications in variable sequence contexts will modulate the nanopore current.  To accomplish this we have generated training sets to characterize N6-methyladenosine,

focusing on the METTL3 motif (GGm6ACU). We identified variable current signatures in regions with known modifications, even identifying isoform specific modification patterns.

 

Examining poly(A) tail lengths, we have applied software (nanopolish) to call poly-A tails from our native RNA sequencing data. Using this software we measured gene specific and even isoform specific poly(A) tail lengths.  As expected, we measured a clear difference between mitochondrial transcripts with a shorter poly(A) tail length than nuclear transcripts. Specifically we found different isoforms of the same gene with different poly(A) tail lengths, with isoforms with retained introns typically having a higher average tail length than isoforms where the intron was spliced out.

Authors:
  • Roham Razaghi
    Author Email
    rrazagh1@jhu.edu
    Institution
    Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD

Methods and Tools for MeRIP-seq and nanopore detection of m6A(m) changes

Speaker:
Track:

Previous studies have reported that various stimuli, including viral infection, cancer, and heat shock, lead to changes to m6A methylation of mRNAs from global increases or decreases in m6A to shifts in the distribution of methylation over transcripts or changes at specific loci. Reported estimates of m6A gains and losses range from zero to thousands of sites, depending on the stimulus and analysis methods. Most studies map m6A using methylated RNA immunoprecipitation sequencing (MeRIP-seq). Methylated regions are identified through peaks in transcript coverage from the immunoprecipitated fraction compared to an input fraction. However, for any two given studies looking at similar conditions, we found peak overlap varied from ~30 to 60% , independent of the cell lines studied. Although we verified appropriate statistical methods to detect changes in peaks using positive and negative controls, we were unable to validate consistent changes across studies of similar experimental interventions. Eventually, single-molecule methods may help in these challenges, and as such, we also present evidence of m6A within nature RNA nanopore molecules with a new tool (EpinNano).

Authors:
  • Christopher E. Mason; Alexa B.R. McIntyre; Nandan S. Gokhale; Leandro Cerchietti; Stacy M. Horner
    Author Email
    christopher.e.mason@gmail.com
    Institution
    Cornell Wiell School of Medicine

Signal analysis of nanopore RNA sequencing to interrogate poly(A) tails and post-transcriptional modifications

Speaker: Roham Razaghi
Track:

Although development of high throughput RNA sequencing technologies has allowed significant advances in our understanding of transcriptome complexity and regulation, certain questions remain intractable with conventional short read cDNA sequencing. Native RNA sequencing technology can address these difficult questions; including identity and frequency of splice variants, full-length poly(A) tail assessment, base modifications and transcript haplotyping. To accomplish this, we leveraged our extensive consortia-generated GM12878 native RNA dataset to showcase this exciting technology and the promise it holds for the direct and simultaneous interrogation of these RNA features. 
We have applied tools to directly interrogate the raw electrical current data from nanopore sequencing to examine these questions.  Specifically, we have begun to empirically establish how different modifications in variable sequence contexts will modulate the nanopore current.  To accomplish this we have generated training sets to characterize N6-methyladenosine, focusing on the METTL3 motif (GGm6ACU). We identified variable current signatures in regions with known modifications, even identifying isoform specific modification patterns. 
Examining poly(A) tail lengths, we have applied software (nanopolish) to call poly-A tails from our native RNA sequencing data. Using this software we measured gene specific and even isoform specific poly(A) tail lengths.  As expected, we measured a clear difference between mitochondrial transcripts with a shorter poly(A) tail length than nuclear transcripts. Specifically we found different isoforms of the same gene with different poly(A) tail lengths, with isoforms with retained introns typically having a higher average tail length than isoforms where the intron was spliced out. 

Authors:
  • Roham Razaghi
    Author Email
    rrazagh1@jhu.edu
    Institution
    Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD

Platinum Sponsor Talk: New Options for Methylome Analysis and RNA Depletion

Speaker: Fiona Stewart
Track:

As the range of samples sequenced expands, the demands on NGS sample preparation increase, and improved options are required for applications including methylome analysis and transcriptome analysis.

Accurate identification of 5mC and 5hmC in DNA increases insight into potential gene regulatory mechanisms. Bisulfite sequencing is traditionally used to detect methylated C's, but the chemical bisulfite reaction damages DNA, and libraries with high GC-bias, and enriched for methylated regions are produced. We have developed an enzyme-based approach for methylation detection, NEBNext Enzymatic Methyl-seq (EM-seq), that minimizes DNA damage. EM-seq libraries have longer inserts, lower duplication rates, a higher percentage of mapped reads and less GC bias compared to bisulfite-converted libraries.

In whole-transcriptome sequencing, highly expressed transcripts with minimal biological interest can dominate readouts, masking detection of more informative lower abundance transcripts. For applications where the full spectrum of RNA is of interest or where poly(A) tails are not present, a method for depletion of abundant RNAs is required. Probe sets for the NEBNext RNaseH-based depletion method are being expanded to remove adult, fetal and embryonic hemoglobin transcripts from blood samples (in which globin can constitute up to ~60% of total mRNA transcripts) and from bacteria, as well as development of a custom probe design tool.

Authors:
  • Fiona Stewart
    Author Email
    stewart@neb.com
    Institution
    New England Biolabs