Read Quality Assessment & Improvement. J Fass UCD Genome Center Bioinformatics Core Monday June 16, 2014
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1 Read Quality ssessment & Improvement J Fass UCD Genome Center Bioinformatics Core Monday June 16, 2014
2 Error modes Each technology has unique error modes, depending on the physico-chemical processes involved in the whole sequencing life cycle (not just base-calling step).
3 Illumina 3'-end noise
4 Illumina 3'-end noise
5 Illumina 3'-end noise
6 Illumina 3'-end noise
7 Illumina 3'-end noise
8 Illumina 3'-end noise
9 Illumina 3'-end noise
10 454, Ion Torrent homopolymer runs Nucleotides are not "terminated," so homopolymer runs add bases all at the same time. n absolute level of uncertainty / noise in the fluorescence signal will have a greater proportional effect on longer homopolymer runs. I.e. 4 's vs 5 's is a 25% difference... 8 's vs 9 's is a 12% difference
11 PacBio errors C G T T G C C G T T G C G T T G C G T T G C G T T G C G T T G false peak identified true peak missed
12 The reads are (not) alright... Contaminating sequence within reads adapters Poor quality sequence substitution, indel errors Sample contamination Chimerism in library
13 dapter contamination
14 dapter contamination Older "in-line" or "homebrew" adapters can be added to one or both ends of DN library fragments. Tools like Sabre (Nik Joshi) can recognize these, separate reads into different files, and remove barcode bases.
15 dapter contamination The problem is heterogeneous fragment sizes, resulting from any of the current library preparation techniques. ll libraries will contain DN fragments of variable size.
16 dapter contamination Contamination is the result of the sequencer reading through a short read, into adapter sequence that didn't come from your sample!
17 dapter contamination Where can you find out adapter sequences? Google "github ucdavis-bioinformatics" Check Seqanswers.com Contact Illumina, PacBio, etc. for "tech notes" specifying the library prep primer / adapter sequences (not always that clear to work out). Find them in your data. Scythe "*_adapters.fa"
18 Base qualities
19 Base quality in the FSTQ format
20 FSTQ - Pop Quiz! 1. What does a quality character of ";" mean? 2. In Sanger (standard) FSTQ, which SCII character would I use to indicate that I'm absolutely sure that I'm wrong about a particular base? 3. If a particular 40 bp read from a run analyzed with Illumina Pipeline 1.6 (phred + 64) had consistent quality characters of "J", how many errors should you expect in the read?
21 FSTQ - Base order / read orientation n "F/R" pair, or "innies"
22 Back to contamination / quality issues
23 Back to contamination / quality issues
24 (side note - "file" issues) Do your FSTQ files begin and end with the same IDs? Incomplete downloads, accidental sorting, different trimming, etc. can get your forward and reverse read files out of sync with each other.
25 Sabre
26 Scythe
27 Sickle
28 File Formats FST FSTQ SFF (Standard Flowgram Format) HDF5
29 File Formats Illumina FSTQ, BM 454, Ion Torrent(?) SFF FSTQ, FST + QUL sff_extract script, Roche's GS-Tools PacBio HDF5 ("*.bas.h5") FSTQ, FST + QUL Secondary_nalysis_Results ("filtered_subreads.fastq")
30 Error Correction Illumina k-mer spectrum - based Quake CORL PacBio PBcR (Koren 2012 Nature Biotech)... in SMRT-nalysis tools
31 Error Correction (PBcR)
32 Data "grooming"
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