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LondonCalling2018_TranscriptomicBreakout

 LondonCalling2018_TranscriptomicBreakout

Breakout talk in transcriptomics given at the London Calling Conference in 2018. Overviews initial attempts at capturing post-transcriptional regulation in dRNA sequencing of C. elegans across its development.

Nathan Roach

May 25, 2018
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  1. @NanoporeConf | #NanoporeConf Measuring the transcriptome of the C. elegans

    lifecycle using direct RNA sequencing Nathan Roach Johns Hopkins University [email protected]
  2. 2 @NanoporeConf | #NanoporeConf Post-transcriptional regulation of RNA transcripts pre-mRNA

    Alt. Splicing Alt. PAS / 3’ UTR Capping Poly(A) tail length AAA AAAAAAA Regulatory Binding Sites
  3. 5 @NanoporeConf | #NanoporeConf Summary of sequencing data Sample Tech

    Replicate # of Bases (Millions) # of Reads Avg Per Base Quality L1 1 179 196,311 11.4 2 512 522,582 11.8 L2 1 60 65,500 11.3 2 590 600,111 12.0 L3 1 185 206,349 11.5 2 477 501,972 12.1 L4 1 255 275,559 11.5 2 173 194,335 11.7 Young Adult 1 933 832,741 11.4 2 81 79,640 12.2 Male 1 329 345,420 11.4
  4. 8 @NanoporeConf | #NanoporeConf Quantifying transcriptome capture Adapted from Burser

    & Gugio. Genomics. 1996. Illumina Annotation Nanopore Annotation TN FN TN TN TP TP FP FN FN Sensitivity = #$ #$%&' Specificity = #$ #$%&$ TP = True Positive FP = False Positive TN = True Negative FN = False Negative
  5. 10 @NanoporeConf | #NanoporeConf Combinatorial splicing captured by nanopore reads

    unc-32 Annotation Young Adult Reads 43% / 7% / 50% 3% / 97%
  6. 11 @NanoporeConf | #NanoporeConf Combinatorial splicing captured by nanopore reads

    22% / 33% / 44% L4 Reads 66% / 33% unc-32 Annotation
  7. 12 @NanoporeConf | #NanoporeConf 3’UTR choice can be resolved with

    nanopore reads L1 Reads Male Reads 9631.0 9630.8 9630.6 9630.4 Kb ubc-7 Annotation 9631.0 9630.8 9630.6 9630.4 Kb
  8. @NanoporeConf | #NanoporeConf Estimating poly(A) tail lengths with the nanopolish

    suite https://github.com/jts/nanopolish/tree/polya-merged (Experimental) 2
  9. 14 @NanoporeConf | #NanoporeConf Poly(A) tail length can be estimated

    from raw signal Garalde et al. Nature Methods. 2018.
  10. 15 @NanoporeConf | #NanoporeConf Estimated Poly(A) tail lengths Lima et

    al. Nat Struct Mol Biol. 2017. https://github.com/jts/nanopolish/tree/polya-merged 0.00 0.01 0.02 0.03 50 100 150 200 Estimated Bases Proportion of mapped reads PolyA Len Distribution
  11. 16 @NanoporeConf | #NanoporeConf Poly(A) Length vs Isoform NR_002368 NM_001026085

    NM_001026084 NM_001026083 https://github.com/jts/nanopolish/tree/polya-merged 0 40 80 120 NM_001026083 NM_001026084 NM_001026085 NR_002368 rpl−3 Isoforms Estimated Poly(A) Length Isofor NM_ NM_ NM_ NR_ 1.6 Kb
  12. 17 @NanoporeConf | #NanoporeConf Poly(A) of eft-3 across developmental stages

    0 40 80 120 L1 L2 L3 L4 ML YA stage eft−3 polyA tail lengths stage L1 L2 L3 L4 ML YA Male Adult
  13. 19 @NanoporeConf | #NanoporeConf © 2018 Oxford Nanopore Technologies. All

    rights reserved. MinION, Flongle, GridION, PromethION and VolTRAX are currently for research use only. Acknowledgements Kim Lab: John Kim Amelia Alessi Charlotte Choi Nicita Mehta Himani Galagali Jessie Kirshner Mindy Clark Greg Fuller Rebecca Tay Oxford Nanopore Technologies Taylor Lab: James Taylor Mallory Freeberg Mike Sauria Enis Afgan Peter DeFord Min Hyung Cho Boris Brenerman Timp Lab: Winston Timp Rachel Workman Norah Sadowski