• Document: small RNA-seq data analysis
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Long and short/small RNA-seq data analysis GEF5,  4.9.2015 Sami  Heikkinen,  PhD,  Dos. UEF // University of Eastern Finland Topics 1.  RNA-seq in a nutshell 2.  Long vs short/small RNA-seq 3.  Bioinformatic analysis work flows UEF // University of Eastern Finland GEF5 / Heikkinen S / 4.9.2015 2 RNA-seq in a nutshell UEF // University of Eastern Finland GEF5 / Heikkinen S / 4.9.2015 3 Bench RNA-seq Define problem project Consult bioinformatician (et al)! Planning Get ethical permits work flow Select/get samples (groups, N) Define sequencing strategy Extract RNA Execution Generate sequencing libraries Sequence Bed side Perform QC Individualized Preprocess, align, analyze / test treatment? Bioinformatics Genetic risk? Summarize, visualize Interpret! etc! UEF // University of Eastern Finland GEF5 / Heikkinen S / 4.9.2015 4 Next Generation Sequencing (NGS) •  =deep sequencing (Fin: syväsekvensointi) •  “counting” applications –  the count of reads aligning to a genomic location matter (the most) –  e.g. ChIP-seq, RNA-seq, many others •  “qualitative” applications –  the sequence itself matters –  e.g. whole genome / targeted / exome sequencing d Nature  Reviews  in   Genetics Annu.  Rev.  Anal.  Chem,  Mardis  2013 Me>ger  2010 UEF // University of Eastern Finland GEF5 / Heikkinen S / 4.9.2015 5 Sample barcoding Sample  1 Sample  2 Barcoded   e.g.  ATCACG sequencing   libraries Linkers   DNA  fragment Linkers   &   &   Sequencing adapters adapters •  allows for multiplexing -  take benefit of the modern high-capasity sequencers: All  reads ~200 million reads per one run on “old” Illumina HiSeq -  recent versions up to 20x that! -  typically up to 48 or even 96 barcodes De-­‐‑bar   coded   reads Sample  1 Sample  2 UEF // University of Eastern Finland GEF5 / Heikkinen S / 4.9.2015 6 Read length Reads Fragments • most counting applications: 50 bp Target  mRNA –  genome sequencing: 100 – 600 bp 100  bp • long RNA-seq –  only measure gene expression levels (etc)? 50 bp OK –  interested in alternative splicing? Need 100+ bp! Sequence  reads 50  bp 100  bp Sequenced  fragment • short/small RNA-seq Target  mRNA Exon  1 Exon  2 Exon  3 –  e.g. mature miRNAs ~22 nt  ~40 bp UEF // University of Eastern Finland

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