1. Next-generation sequence analysis Gabor T. Marth Boston College Biology Department PSB 2008 January 4-8. 2008

2. Read length and throughput read length bases per machine run 10 bp1,000 bp100 bp 100 Mb 10 Mb 1Mb 1Gb Illumina/Solexa, AB/SOLiD short-read sequencers ABI capillary sequencer 454 pyrosequencer (20-100 Mb in 100-250 bp reads) (1-4 Gb in 25-50 bp reads)

3. DNA ligationDNA base extension Church, 2005 Sequencing chemistries

4. Template clonal amplification Church, 2005

5. Massively parallel sequencing Church, 2005

6. Features of NGS data Short sequence reads –100-200bp –25-35bp (micro-reads) Huge amount of sequence per run –Up to gigabases per run Huge number of reads per run –Up to 100’s of millions Higher error as compared with Sanger sequencing –Error profile different to Sanger

7. Current and future application areas Genome re-sequencing: somatic mutation detection, organismal SNP discovery, mutational profiling, structural variation discovery De novo genome sequencing Short-read sequencing will be (at least) an alternative to micro-arrays for: DNA-protein interaction analysis (CHiP-Seq) novel transcript discovery quantification of gene expression epigenetic analysis (methylation profiling) DEL SNP reference genome

8. Fundamental informatics challenges 1. Interpreting machine readouts – base calling, base error estimation 2. Dealing with non- uniqueness in the genome: resequenceability 3. Alignment of billions of reads

9. Informatics challenges (cont’d) 5. Data visualization 4. SNP and short INDEL, and structural variation discovery 6. Data storage & management

10. Challenge 1. Base accuracy and base calling machine read-outs are quite different read length, read accuracy, and sequencing error profiles are variable (and change rapidly as machine hardware, chemistry, optics, and noise filtering improves) what is the instrument-specific error profile? are the base quality values satisfactory? (1) are base quality values accurate? (2) are most called bases high-quality?

11. 454 pyrosequencer error profile multiple bases in a homo-polymeric run are incorporated in a single incorporation test  the number of bases must be determined from a single scalar signal  the majority of errors are INDELs error rates are nucleotide-dependent

12. 454 base quality values the native 454 base caller assigns too low base quality values

13. PYROBAYES: determine base number data likelihoods priors posterior base number probability New 454 base caller:

14. PYROBAYES: base calls and quality values call the most likely number of nucleotides produce three base quality values: QS (substitution) QI (insertion) QD (deletion)

15. PYROBAYES: Performance better correlation between assigned and measured quality values higher fraction of high-quality bases

16. Illumina/Solexa base accuracy error rate grows as a function of base position within the read a large fraction of the reads contains 1 or 2 errors

17. Illumina/Solexa base accuracy (cont’d) Actual base accuracy for a fixed base quality value is a function of base position within the read (i.e. there is need for quality value calibration) Most errors are substitutions  PHRED quality values work

18. 3’5’ N N N T G z z z 3’5’ N N N G A z z z 3’5’ N N N A T z z z 2-base, 4-color: 16 probe combinations ●4 dyes to encode 16 2-base combinations ●Detect a single color indicates 4 combinations & eliminates 12 ●Each color reflects position, not the base call ●Each base is interrogated by two probes ●Dual interrogation eases discrimination –errors (random or systematic) vs. SNPs (true polymorphisms) ACGT A C G T 2 nd Base 1 st Base 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 AB SOLiD System dibase sequencing

19. The decoding matrix allows a sequence of transitions to be converted to a base sequence, as long as one of two bases is known. ACGT A C G T 2 nd Base 1 st Base 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 AA AC AC AA AG AT AA AG AG CC CA CA CC CT CG CC CT CT GG GT GT GG GA GC GG GA GA TT TG TG TT TC TA TT TC TC A A C A A G C C T C C C A C C T A A G A G G T G G A T T C T T T G T T C G G A G 1 00 1 23022 1 00 1 23022 4 Possible Sequences Converting dibase (color) into base calls

20. Reference Alignment to reference in “color-space” Working in color space: –Reverse-complementation becomes simply reverse –Apply color transition rules to remove measurement errors from partial assemblies –If reference of Sanger reads are combined, translate to color space

21. A C G G T C G T C G T G T G C G T A C G G T C G C C G T G T G C G T A C G G T C G T C G T G T G C G T No change SNP Measurement error SOLiD error checking code (I)

22. G T C encodes 3 Possible Changes in the Middle Base 3 Possible Changes in Dibase Encoding G A C encodes G C C encodes G G C encodes Allowed Transitions Only Some Transitions Indicate a SNP in sample SOLiD error checking code (II)

23. A C G G T C G T C G T G T G C G T A C G G T C - T C G T G T G C G T A C G G T C - - C G T G T G C G T A C G G T C G T C G T G T G C G T Invalid adjacent 1 base deletion 2 base deletion SOLiD error checking code (III)

24. SOLiD di-base sequencing accuracy and QV

25. Challenge 2. Resequenceability Reads from repeats cannot be uniquely mapped back to their true region of origin RepeatMasker does not capture all micro-repeats, i.e. repeats at the scale of the read length Near-perfect micro-repeats can be also a problem because we want to align reads even with a few sequencing errors and / or SNPs

26. Repeats at the fragment level “base masking” “fragment masking”

27. Fragment level repeat annotation bases in repetitive fragments may be resequenced with reads representing other, unique fragments  fragment-level repeat annotations spare a higher fraction of the genome than base-level repeat masking

28. Find perfect and near-perfect micro-repeats Hash based methods (fast but only work out to a couple of mismatches) Exact methods (very slow but find every repeat copy) Heuristic methods (fast but miss a fraction of the repeats)

29. Challenge 3. Read alignment and assembly resequencing requires reference sequence-guided read alignment to align billions of reads the aligner has to be fast and efficient INDEL errors require gapped alignment individually aligned reads must be “assembled” together has to work for every read type (short, medium-length, and long reads) must tolerate sequencing errors and SNPs must work with both base-level and fragment-level repeat annotations transcribed sequences require additional features e.g. splice-site aware alignment capability most frequently used tools: BLAT (only pair-wise), SSAHA (pair-wise), MAQ (pair-wise and assembly), ELAND (pair-wise), MOSAIK (pair-wise and assembly, gapped)

30. MOSAIK: method Step 1. initial short-hash based scan for possible read locations Step 2. evaluation of candidate read locations with SW method

31. MOSAIK – performance Solexa read alignments to C. elegans genome: 100 million reads aligned in 95 minutes 18,000 reads / second 454 reads to Pichia (yeast-size) genome GS20: 2,000 reads / second FLX: 300 reads / second Solexa read alignments to masked human genome: 40 seconds for 1 million reads 18,000 reads / second 5.5 GB RAM used (more for longer initial hash sizes)

32. MOSAIK: co-assembling different read types ABI/cap. 454/FLX Illumina 454/GS20

33. Challenge 4. Polymorphism discovery shallow and deep read coverage most candidates will never be “checked”  only very low error rates are acceptable we updated PolyBayes to deal with new read types made the new software (PBSHORT) much more efficient

34. Structural variation discovery copy number variations (deletions & amplifications) can be detected from variations in the depth of read coverage structural rearrangements (inversions and translocations) require paired-end read data

35. Challenge 5. Data visualization 1.aid software development: integration of trace data viewing, fast navigation, zooming/panning 2.facilitate data validation (e.g. SNP validation): co-viewing of multiple read types, quality value displays 3.promote hypothesis generation: integration of annotation tracks

36. Challenge 6. Massive data volumes Short-read format working group ssrformat@ubc.ca (Asim Siddiqui, UBC) Assembly format working group Boston College http://assembly.bc.edu two connected working groups to define standard data formats

37. Next-generation sequencing software http://bioinformatics.bc.edu/marthlab/Mosaik http://sourceforge.net/projects/maq/ Machine manufacturers’ sites plus third- party developers’ sites, e.g.:

38. Applications in various discovery projects 1. SNP discovery in shallow, single-read 454 coverage (Drosophila melanogaster) 2. Mutational profiling in deep 454 data (Pichia stipitis) 3. SNP and INDEL discovery in deep Illumina / Solexa short-read coverage (Caenorhabditis elegans) (image from Nature Biotech.)

39. SNP calling in single-read 454 coverage collaborative project with Andy Clark (Cornell) and Elaine Mardis (Wash. U.) goal was to assess polymorphism rates between 10 different African and American melanogaster isolates 10 runs of 454 reads (~300,000 reads per isolate) were collected key informatics question: can we detect SNPs with high accuracy in low-coverage, survey-style 454 reads aligned to finished reference genome sequence? DNA courtesy of Chuck Langley, UC Davis reads were base-called with PyroBayes and aligned to the 180Mb reference melanogaster genome sequence with Mosaik  0.16 x nominal read coverage  most reads are singletons SNP detection with PolyBayes

40. SNP calling success rates iso-1 reference 46-2 454 read 46-2 ABI reads (2 fwd + 2 rev) 92.9 % validation rate (1,342 / 1,443) single-read coverage: 92.9% (1,275 / 1,372 ) double-read coverage: 94.3% (67 / 71) 2.0% missed SNP rate (25 / 1247) single-read coverage: 2.12% (25 / 1176) double-read coverage: 0% (0 / 59)

41. Genome variation in melanogaster isolates 658,280 SNPs discovered among all 10 lines. Nucleotide diversity Ѳ ≈ 5x10 -3 (1 SNP / 200 bp) between each line and reference (in line with expectations). 20.2% (133,264 sites) polymorphic among two or more lines. The 1 SNP / 900 bp nominal density is sufficient for high-resolution marker mapping

42. SNP calling in short-read coverage C. elegans reference genome (Bristol, N2 strain) Pasadena, CB4858 (1 ½ machine runs) Bristol, N2 strain (3 ½ machine runs) goal was to evaluate the Solexa/Illumina technology for the complete resequencing of large model-organism genomes 5 runs (~120 million) Illumina reads from the Wash. U. Genome Center, as part of a collaborative project lead by Elaine Mardis, at Washington University primary aim was to detect polymorphisms between the Pasadena and the Bristol strain

43. Polymorphism discovery in C. elegans SNP calling error rate very low: Validation rate = 97.8% (224/229) Conversion rate = 92.6% (224/242) Missed SNP rate = 3.75% (26/693) SNP INS INDEL candidates validate and convert at similar rates to SNPs: Validation rate = 89.3% (193/216) Conversion rate = 87.3% (193/221) MOSAIK aligned / assembled the reads (< 4 hours on 1 CPU) PBSHORT found 44,642 SNP candidates (2 hours on our 160-CPU cluster) SNP density: 1 in 1,630 bp (of non-repeat genome sequence)

44. Mutational profiling: deep 454/Illumina/SOLiD data collaboration with Doug Smith at Agencourt Pichia stipitis converts xylose to ethanol (bio-fuel production) one mutagenized strain had especially high conversion efficiency determine where the mutations were that caused this phenotype we resequenced the 15MB genome with 454 Illumina, and SOLiD reads 14 true point mutations in the entire genome Pichia stipitis reference sequence Image from JGI web site

45. Mutational profiling: comparisons TechnologyCoverageNominal coverageFPFNTotal error 454/FLX2 runs12.9x101 454/FLX1 run9.8x617 Illumina7 lanes53.5x000 Illumina3 lanes23.4x000 Illumina2 lanes15.6x202 Illumina1 lane7.6x222 SOLiD-30.0X000 SOLiD-20.0X000 SOLiD-10.0X000 SOLiD-8.0X044 SOLiD-6.0X066

46. Informatics of transcriptome sequencing measuring gene expression levels by sequence tag counting requires SAGE informatics-like approaches novel transcript discovery Inferred Exon 1Inferred Exon 2 Inferred Exon 1Inferred Exon 2 new genes & exons novel transcripts in known genes

47. Protein-DNA interactions: CHiP-Seq Protein-bound DNA fragments are isolated with chromatin immunoprecipitation (ChIP) and then sequenced (Seq) on a high- throughput sequencing platform. Sequences are mapped to the genome sequence with a read alignment program. Regions over-represented in the sequences are identified. Johnson et al. Science, 2007

48. Protein-DNA interactions: CHIP-SEQ Mikkelsen et al. Nature 2007. ChIP-Seq scales well for simultaneous analysis of binding sites in the entire genome.