Illumina Read QC. UCD Genome Center Bioinformatics Core Monday 29 August 2016

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1 Illumina Read QC UCD Genome Center Bioinformatics Core Monday 29 August 2016

2 QC should be interactive

3 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). Improving reads will work better if the assumptions made by the remediation tools match the source(s) of error. How do you know? Trial and error? Read QC is experimental, just like bench science.

4 Illumina read problems Contaminating sequence within reads adapters adapter dimers Poor quality and/or wrong sequence substitution, insertion / deletion ( indel ) errors Sample contamination Chimerism in library Sampling bias

5 Illumina errors Illumina errors are biased - they occur after some sequence motifs (not well addressed by any tools currently, IMO), and predominantly at the 3 -ends of reads.

6 Illumina - 3 -end errors

7 Illumina - 3 -end errors

8 Illumina - error rates Overall Illumina error rate ~ 0.1-1% Of that, 99% are substitutions, 1% are insertions / deletions ( indels )

9 Quality Remediation: Sickle

10 Adapter contamination

11 Adapter contamination Older "in-line" or "homebrew" adapters can be added to one or both ends of DNA library fragments. Tools like Sabre (Nik Joshi) can recognize these, separate reads into different files, and remove barcode bases.

12 Adapter contamination The problem is heterogeneous fragment sizes, resulting from any of the current library preparation techniques. All libraries will contain DNA fragments of variable size.

13 Adapter contamination Contamination is the result of the sequencer reading through a short read, into adapter sequence that didn't come from your sample!

14 Adapter contamination Where can you find out adapter sequences? Google "github ucdavis-bioinformatics", look for Scythe, look for "*_adapters.fa" 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.

15 Adapter contamination >TruSeq_forward_contam AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC[8bp index]atctcgtatgccgtcttctgcttgaaaaa >TruSeq_reverse_contam AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT[8bp index]gtggtcgccgtatcattaaaaa >Nextera_forward_contam CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[8bp index]atctcgtatgccgtcttctgcttg >Nextera_reverse_contam CTGTCTCTTATACACATCTGACGCTGCCGACGA[8bp index]gtgtagatctcggtggtcgccgtatcatt >TruSeq_SmallRNA_forward_contam TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC[6bp adapter]atctcgtatgccgtcttctgcttg >TruSeq_SmallRNA_reverse_contam GATCGTCGGACTGTAGAACTCTGAACCTGTCG Also note small RNA trimming instructions here: find mirna on page

16 Adapter Removal: Scythe

17 Error Correction Paired-read overlap ( read merging, paired read assemblers ) FLASH PEAR PANDAseq Correct bases in overlapping region; output a single read No merging / correction possible; output pair of reads Correct in overlapping region; trim overhangs (adapter); output single read

18 Questions?

19 Illumina - 3 -end errors (glass substrate)

20 Illumina - 3 -end errors (glass substrate)

21 Illumina - 3 -end errors 5 -CTCTTCCGATCT <-- add sequencing primers 5 -CTCTTCCGATCT 5 -CTCTTCCGATCT 5 -CTCTTCCGATCT 5 -CTCTTCCGATCT 5 -CTCTTCCGATCT (glass substrate) 5 -CTCTTCCGATCT 5 -CTCTTCCGATCT

22 Illumina - 3 -end errors 5 -CTCTTCCGATCTC <-- cycle 1 5 -CTCTTCCGATCTC 5 -CTCTTCCGATCTC 5 -CTCTTCCGATCTC 5 -CTCTTCCGATCTC 5 -CTCTTCCGATCTC (glass substrate) 5 -CTCTTCCGATCTC 5 -CTCTTCCGATCTC

23 Illumina - 3 -end errors 5 -CTCTTCCGATCTCT <-- cycle 2 5 -CTCTTCCGATCTCT 5 -CTCTTCCGATCTCT 5 -CTCTTCCGATCTCT 5 -CTCTTCCGATCTCT 5 -CTCTTCCGATCTCT (glass substrate) 5 -CTCTTCCGATCTCT 5 -CTCTTCCGATCTCT

24 Illumina - 3 -end errors 5 -CTCTTCCGATCTCTC <-- cycle 3 5 -CTCTTCCGATCTCTC 5 -CTCTTCCGATCTCTC 5 -CTCTTCCGATCTCTC 5 -CTCTTCCGATCTCTC 5 -CTCTTCCGATCTCTC (glass substrate) 5 -CTCTTCCGATCTCTC 5 -CTCTTCCGATCTCTC

25 Illumina - 3 -end errors 5 -CTCTTCCGATCTCTCTGCGCTTGAGAG in phase 5 -CTCTTCCGATCTCTCTGCGCTTGAGAG in phase 5 -CTCTTCCGATCTCTCTGCGCTTGAGAGA pre-phasing (+1) 5 -CTCTTCCGATCTCTCTGCGCTTGAGAG in phase 5 -CTCTTCCGATCTCTCTGCGCTTGAGAG in phase 5 -CTCTTCCGATCTCTCTGCGCTTGAGAG in phase (glass substrate) 5 -CTCTTCCGATCTCTCTGCGCTTGAGA post-phasing (-1) 5 -CTCTTCCGATCTCTCTGCGCTTGAGAG in phase

26 Illumina - 3 -end errors Cycle 1 # of molecules A T C G True cycle offset (pre- / post-phasing events)

27 Illumina - 3 -end errors Cycle 15 # of molecules stochastic variability A T C G Process Error

28 Illumina - 3 -end errors Intensity = A T C G Measurement Error

29 Illumina - 3 -end errors Measurement Error