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![[I0D51A] Bioinformatics: High-Throughput Analysis
Next-generation sequencing. Part 1: Technologies
Prof Jan Aerts
Faculty of Engineering - ESAT/SCD
jan.aerts@esat.kuleuven.be
TA: Alejandro Sifrim (alejandro.sifrim@esat.kuleuven.be)
1](https://image.slidesharecdn.com/i0d51a20110506-110505140616-phpapp02/75/Next-generation-sequencing-course-part-1-technologies-1-2048.jpg)
Uploaded byJan Aerts
Next-generation sequencing course, part 1: technologies
This document provides an overview of next-generation sequencing technologies and their applications. It discusses genome enrichment techniques to isolate targeted regions for sequencing. It also describes template preparation methods like emulsion PCR and solid-phase amplification. Finally, it reviews various sequencing platforms like Illumina, SOLiD, 454 and details the sequencing and imaging processes. There are exercises proposed to work with sequencing data files in Galaxy.
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Next-generation sequencing course, part 1: technologies
- 1. [I0D51A] Bioinformatics: High-ThroughputAnalysis Next-generation sequencing. Part 1: Technologies Prof Jan Aerts Faculty of Engineering - ESAT/SCD jan.aerts@esat.kuleuven.be TA: Alejandro Sifrim (alejandro.sifrim@esat.kuleuven.be) 1
- 2. Announcements May 27th (9am-noon):evaluation open book 2
- 3. Note to self... Uploads_1_sequence.txt and s_2_sequence.txt to Galaxy first... 3
- 4. Overview • linux refresher(6/5) • next-generation sequencing technologies and applications (6/5) • sequence mapping (13/5) • variant calling - SNPs (20/5) • variant calling - structural variation (20/5) 4
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- 9. First vs secondgeneration sequencing Sanger sequencing (1st gen) 2nd/next gen sequencing Shendure & Ji, 2008 9
- 10. Paired-end sequencing Korbel et al, 2007 10
- 11. General approaches • 2ndgeneration: clonally amplified single molecules • Roche 454 pyrosequencing • Illumina Genome Analyzer -> HiSeq: reversible terminator technology • ABI SOLiD: ligation-based extension • Next-next-generation/3rd generation: true single molecule • Helicos: Heliscore • Pacific Biosciences: SMRT 11
- 12. Mardis, 2011 12
- 13. Steps genome enrichment template preparation sequencing and imaging data analysis 13
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- 16. What? Only sequence relevantparts of the genome instead of whole genome, e.g.: • specific Mb-scale regions known to be involved in particular disease (e.g. based on GWAS) • specific candidate genes belonging to disease pathway • exome (= all exons) => how to isolate these from non-target sequence? “pulldown” 16
- 17. Pulldown: on-array Turner et al, 2009 17
- 18. Pulldown: in-solution Turner et al, 2009 18
- 19. Performance metrics • fold-enrichment:ratio of abundance of target sequences post-enrichment vs pre-enrichment • capture specificity: fraction of sequence reads that map to target • uniformity: relative abundance of individual targets after enrichment • completeness: fraction of target bases detectably captured 19
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- 21. Problem: most imagingsystems not designed to detect single fluorescent event => need amplified templates Aim: to produce a representative, non-biased source of nucleic acid material from the genome under investigation => population of identical templates Steps: 1. shear DNA 2. amplify templates Options: emulsion PCR (emPCR) or solid phase amplification 21
- 22. Amplification by emulsionPCR emulsion = mixture of two or more immiscible (unblendable) liquids; e.g. mayonnaise, vinaigrette emPCR: thousands of microreactors/micro-eppendorfs one bead + one DNA molecule per microreactor => PCR to 1000s of copies 22
- 23. Williams et al,2006 Metzker et al, 2010 23
- 24. Solid-phase amplification http://bit.ly/6JYIUz http://www.youtube.com/watch?v=77r5p8IBwJk&NR=1 Metzker et al, 2010 24
- 25. C. Sequencing andimaging 25
- 26. Sequencing and imaging Technologies: 1.cyclic reversible termination 2. sequencing by ligation 3. pyrosequencing 4. real-time sequencing 26
- 27. Cyclic reversible termination DNAsynthesis is terminated after adding single nucleotide start/stop/start/stop/start/stop/... Illumina: 4-colour sequencing result sequencing steps Metzker et al, 2010 27
- 28. Helicos: 1-colour sequencing steps sequencing result Metzker et al, 2010 Metzker et al, 2010 28
- 29. Sequencing by ligation http://bit.ly/fPh22X sequencing steps 29
- 30.
- 31. Pyrosequencing Metzker et al, 2010 Metzker et al, 2010 31
- 32. Real-time sequencing “ZMW” zero-mode waveguide DNA polymerase “strobe sequencing” 32
- 33. Run time Gb/run Roche 454 8.5 hr 45 Illumina 9 days 35 SOLiD 14 days 50 Helicos 8 days 37 PacBio ? ? 33
- 34. Accuracy - basecalling error • base quality drops along read Sanger > SOLiD > Illumina > 454 > Helicos (“dephasing” within clusters) • base calling errors 34
- 35. Accuracy - homopolymerruns Issue for Roche 454: 39% of errors are homopolymers A5 motifs: 3.3% error rate A8 motifs: 50% error rate Reason: use signal intensity as a measure for homopolymer length 35
- 36.
- 37. Ronaghi, Genome Res11:3-11 (2001) 37
- 38.
- 39. Is it 4?Is it 5? Is it 4? http://mammoth.psu.edu/labPhotos/imageOfFlowgram.jpg 39
- 40. Consensus accuracy Increase accuracyfor SNP calling by increasing coverage: Illumina: 20X SOLiD: 12X 454: 7.4X Sanger: 3X Factors: raw accuracy + read length How deep do you have to sequence? => Poisson distribution: “If you sequence at average of 10X, how much of the genome will be covered at least 5X”? 40
- 41. Bentley et al,Nature 456:53-56 (2008) 41
- 42. FASTQ file format example fasta entries (n=2) “@” + identifier example fastq entries (n=2) sequence “+” + identifier (optional) phred-based quality scores phred quality score encoding Wikipedia 42
- 43. Sequence quality control Isthis good sequence? (essential!) E.g.: using FastQC tool (Babraham Institute, UK; http:// www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) 43
- 44. Sequence quality control per base sequence quality good bad 44
- 45. Sequence quality control per sequence quality scores good bad 45
- 46. Sequence quality control per base sequence content good bad 46
- 47. Sequence quality control per base GC content good bad 47
- 48. Sequence quality control per sequence GC content good bad 48
- 49. Sequence quality control k-mer content good bad 49
- 50.
- 51. Online genome analysis http://galaxy.psu.edu/ “Galaxyallows you to do analyses you cannot do anywhere else without the need to install or download anything. You can analyze multiple alignments, compare genomic annotations, profile metagenomic samples and much much more...” 51
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- 54. Applications of next-generationsequencing 54
- 55. Kahvejian et al,2008 55
- 56. DNA-seq ChIP-seq RNA-seq Kahvejian et al, 2008 50 56
- 57. identify sequence variations DNA-seq ChIP-seq RNA-seq identify pathogens Kahvejian et al, 2008 50 51 57
- 58. Exercises 58
- 59. Try to loginto the server mentioned on Toledo with username and password provided there. There are 2 FASTQ files in /mnt/homes/jaerts/: s_1_sequence.txt and s_2_sequence.txt (= paired ends) • How many sequences are in s_1_sequence.txt? • What encoding was used for the quality score? Illumina? Sanger? • What are the numerical quality scores for the first sequence in s_1_sequence.txt (i.e. 7172283/1)? 59
- 60. • Create anaccount on the Galaxy server • Download s_1_sequence.txt and s_2_sequence.txt from Toledo and upload them into Galaxy. These files are also available on the linux server • Have a look at the contents of s_1_sequence.txt. • Convert quality scores to numeric values for s_1_sequence.txt (“FASTQ Groomer”) • Draw the quality score boxplot for s_1_sequence.txt • Draw the nucleotide distribution chart for s_1_sequence.txt 60
- 61. References Bentley DR etal. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456: 53-59 (2008) Kahvejian A, Quackenbush J & Thompson JF. What would you do if you could sequence everything? Nature Biotechnology 26: 1125-1133 (2008) Korbel JO et al. Paired-end mapping reveals extensive structural variation in the human genome. Science 318: 420-426 (2007) Mardis ER. A decade’s perspective on DNA sequencing technology. Nature 470: 198-203 (2011) Metzker ML. Sequencing technologies - the next generation. Nature Reviews Genetics 11:31-46 (2010) Shendure J & Ji H. Next-generation DNA sequencing. Nature Biotechnology 26:1135-1145 (2008) Turner EH et al. Methods for genomic partitioning. Annual Review of Genomics and Human Genetics 10 (2009) 61