Sequencing 2016
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The document provides an overview of the history and development of DNA sequencing technologies. It discusses early methods like Sanger sequencing and Maxam-Gilbert sequencing. It then summarizes major next-generation sequencing platforms like Illumina, Pacific Biosciences, and Oxford Nanopore. The document also covers sequencing trends, costs, and considerations for choosing a sequencing platform.
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Sequencing 2016
- 1. Surya Saha Sol Genomics Network (SGN) Boyce Thompson Institute, Ithaca, NY suryasaha@cornell.edu // Twitter:@SahaSurya BTI Plant Bioinformatics Course 2016 http://www.acgt.me/blog/2015/3/7/next-generation-sequencing-must-die
- 2. 1953 DNA Structure discovery 1977 2012 Sanger DNA sequencing by chain-terminating inhibitors 1984 Epstein-Barr virus (170 Kb) 1987 Abi370 Sequencer 1995 2001 Homo sapiens (3.0 Gb) 2005 454 Solexa Solid 2007 2011 Ion Torrent PacBio Haemophilus influenzae (1.83 Mb) 2013 Slide concept: Aureliano Bombarely Sequencing over the Ages Illumina Illumina Hiseq X 454 3/29/2016 BTI Plant Bioinformatics Course 2016 2 Pinus taeda (24 Gb) 2014 Nanopore MinION 2015 10X Genomics
- 3. First generation sequencing 3/29/2016 BTI Plant Bioinformatics Course 2016 3 Sanger. Annu Rev Biochem. 1988;57:1-28. Thanks to Nick Loman for the mention
- 4. Maxam-Gilbert method 3/29/2016 BTI Plant Bioinformatics Course 2016 4
- 5. Maxam-Gilbert method 3/29/2016 BTI Plant Bioinformatics Course 2016 5 http://en.wikipedia.org/wiki/File:Maxam- Gilbert_sequencing_en.svg https://www.nationaldiagnostics.com/electrophoresis /article/maxam-gilbert-sequencing
- 6. Sanger method 3/29/2016 BTI Plant Bioinformatics Course 2016 6 Frederick Sanger 13 Aug 1918 – 19 Nov 2013 Won the Nobel Prize for Chemistry in 1958 and 1980. Published the dideoxy chain termination method or “Sanger method” in 1977 http://dailym.ai/1f1XeTB
- 7. Sanger method 3/29/2016 BTI Plant Bioinformatics Course 2016 7 http://en.wikipedia.org/wiki/File:Sanger-sequencing.svg http://en.wikipedia.org/wiki/File: Radioactive_Fluorescent_Seq.jpg
- 8. First generation sequencing • Very high quality sequences (99.999% or Q50) • Very very low throughput 3/29/2016 BTI Plant Bioinformatics Course 2016 8 Run Time Read Length Reads / Run Total nucleotides sequenced Cost / MB Capillary Sequencing (ABI3730xl) 20m-3h 400-900 bp 96 or 384 1.9-84 Kb $2400 http://www.hindawi.com/journals/bmri/2012/251364/tab1/
- 9. Next generation sequencing 3/29/2016 BTI Plant Bioinformatics Course 2016 9
- 10. Use the specific technology used to generate the data – Illumina Hiseq/Miseq/NextSeq – Pacific Biosciences RS I/RS II – Ion Torrent Proton/PGM – SOLiD – Oxford Nanopore 3/29/2016 BTI Plant Bioinformatics Course 2016 10 http://www.acgt.me/blog/2015/3/10/next-generation-sequencing-must- diepart-2
- 11. 454 Pyrosequencing One purified DNA fragment, to one bead, to one read. 3/29/2016 BTI Plant Bioinformatics Course 2016 11 http://www.genengnews.com/ GS FLX Titanium https://mariamuir.com/wp- content/uploads/2013/04/rip.gif
- 12. Illumina 3/29/2016 BTI Plant Bioinformatics Course 2016 12 Output 0.3-15 Gb 20-120 GB 10-1500 GB 900-1800 GB Number of Reads/ Flow cell 25 Million 130-400 Million 300 million – 2.5 Billion 3 Billion Read Length 2x300 bp 2x150 bp 2x250 - 2x125 bp 2x150 bp Cost $99K $250K $740K $10M (10 units) Source: Illumina 2500 3000 4000 500
- 13. Illumina 3/29/2016 BTI Plant Bioinformatics Course 2016 13 Output 0.3-15 Gb 20-120 GB 10-1500 GB 900-1800 GB Number of Reads/ Flow cell 25 Million 130-400 Million 300 million – 2.5 Billion 3 Billion Read Length 2x300 bp 2x150 bp 2x250 - 2x125 bp 2x150 bp Cost $99K $250K $740K $10M (10 units) Source: Illumina 2500 3000 4000 $1000 human genome?? 500
- 14. Illumina 3/29/2016 BTI Plant Bioinformatics Course 2016 14 Mardis 2008. Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402
- 15. Illumina 3/29/2016 BTI Plant Bioinformatics Course 2016 15 Mardis 2008. Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402
- 16. Illumina:TruSeqLongRead 3/29/2016 BTI Plant Bioinformatics Course 2016 16 Voskoboynik eLife 2013;2:e00569
- 17. Pacific Biosciences SMRT sequencing Single Molecule Real Time sequencing 3/29/2016 BTI Plant Bioinformatics Course 2016 17 http://smrt.med.cornell.edu/images/pacbio_library_prep-1.gif RS II Sequel
- 18. Pacific Biosciences SMRT sequencing Error correction methods 3/29/2016 BTI Plant Bioinformatics Course 2016 18 Hierarchical genome-assembly process (HGAP) English et al., PLOS One. 2012 PBJelly
- 19. Pacific Biosciences SMRT sequencing Error correction methods 3/29/2016 BTI Plant Bioinformatics Course 2016 19 PBcRPipeline
- 20. 3/29/2016 Centre for Agricultural Bioinformatics, Pusa 20 Pacific Biosciences SMRT sequencing Read Lengths
- 21. Oxford Nanopore 3/29/2016 Centre for Agricultural Bioinformatics, Pusa 21 https://www.nanoporetech.com/ http://erlichya.tumblr.com/post/66376172948/hands-on- experience-with-oxford-nanopore-minion http://halegrafx.com/vector-art/free-vector-despicable-me-minions/
- 22. 3/29/2016 BTI Plant Bioinformatics Course 2016 22
- 23. Next generation sequencing 3/29/2016 BTI Plant Bioinformatics Course 2016 23 Run Time Read Length Quality Total nucleotides sequenced Cost /MB 454 Pyrosequencing 24h 700 bp Q20-Q30 1 GB $10 Illumina Miseq 27h 2x300bp > Q30 15 GB $0.15 Illumina Hiseq 2500 1 - 10days 2x250bp >Q30 3000 GB $0.05 Ion torrent 2h 400bp >Q20 50MB-1GB $1 Pacific Biosciences 30m - 4h 10kb - >40kb >Q50 consensus >Q10 single 500 - 1000MB /SMRT cell $0.13 - $0.60 http://www.hindawi.com/journals/bmri/2012/251364/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431227 Note: Some figures might be out of date
- 24. Long range scaffolding 3/29/2016 BTI Plant Bioinformatics Course 2016 24
- 25. Hi-C Crosslinking 3/29/2016 BTI Plant Bioinformatics Course 2016 25
- 26. 3/29/2016 BTI Plant Bioinformatics Course 2016 26 http://mms.businesswire.com/media/20150225005296/en /454639/5/GemCodePlatform.jpg • Long read information from short reads using 14bp bar codes • Very low input DNA (ng) and 20 minute library preparation time • 1ng of DNA is split across 100,000 Gel Coated Beads (GEMs) • Chromium instrument for single-cell RNAseq GemCode
- 27. 3/29/2016 BTI Plant Bioinformatics Course 2016 27 http://mms.businesswire.com/media/20150225005296/en /454639/5/GemCodePlatform.jpg GemCode http://www.nature.com/nbt/journal/v34/n3/full/nbt.3432.html
- 28. 3/29/2016 BTI Plant Bioinformatics Course 2016 28 http://www.bionanogenomics.com/technology/why-genome-mapping/
- 29. 3/29/2016 BTI Plant Bioinformatics Course 2016 29 Human MHC map • Sample prep requires very high molecular weight DNA • Nicks at 10 sites / 100kb • Individual molecules are assembles into optical maps • Optical maps and sequences are merged in a hybrid assembly http://www.bionanogenomics.com/technology/why-genome-mapping/
- 30. Many Others.. • Ion Torrent Proton/PGM • Supporting technologies – Nabsys – OpGen – Fluidigm 3/29/2016 BTI Plant Bioinformatics Course 2016 30 http://nextgenseek.com/2012/11/did-you-know-there-are- at-least-14-next-gen-sequence-technology-companies/
- 31. Sequencing Trends 3/29/2016 BTI Plant Bioinformatics Course 2016 31 https://www.google.com/trends/
- 32. 3/29/2016 BTI Plant Bioinformatics Course 2016 32 0 5000 10000 15000 20000 25000 30000 35000 2008 2009 2010 2011 2012 2013 2014 2015 Number of Publications Illumina Pacific Biosciences Roche 454 Ion Torrent -2000 -1000 0 1000 2000 3000 4000 5000 6000 2009 2010 2011 2012 2013 2014 2015 Increase in Number of Publications Illumina Pacific Biosciences Roche 454 Ion Torrent 0.00% 20.00% 40.00% 60.00% 80.00% 100.00% 120.00% 2009 2010 2011 2012 2013 2014 2015 % Increase in Number of Publications Pacific Biosciences Roche 454 Ion Torrent
- 33. Real cost of Sequencing!! Sboner, Genome Biology, 2011 3/29/2016 33BTI Plant Bioinformatics Course 2016
- 34. 3/29/2016 BTI Plant Bioinformatics Course 2016 34 https://genomebiology.biomedcentral.com/articles/10.1186/gb-2011-12-8-125
- 35. So What Sequencer Do I Use?? Microbial genome • Draft genome – Illumina Miseq (100-130X) – Illumina Hiseq (<200X) • Complete genome – Pacific Biosciences (80-100X) • Amplicons (16S, ITS) – Illumina Miseq Eukaryotic genome • Denovo assembly – Pacific Biosciences (70-80X) – Illumina Hiseq (100X+) – 10X Genomics – Bionano • Genotyping (GBS) – Illumina Hiseq • BACs – Pacific Biosciences 3/29/2016 BTI Plant Bioinformatics Course 2016 35 $$$$ ????
- 36. 3/29/2016 BTI Plant Bioinformatics Course 2016 36 The diploid reference genome
- 37. Cornell Sequencing Core • Illumina Hiseq 2500 (Rapid run and High output) • Illumina Miseq • Illumina Nextseq 500 • 10X Genomics GemCode 3/29/2016 BTI Plant Bioinformatics Course 2016 37 http://www.biotech.cornell.edu/brc/g enomics/services/price-list#overlay- context=brc/genomics-facility/next- generation-sequencing $ $ $
- 38. Library Types Single end Pair end (PE, 150-300 bp, Fwd:/1, Rev:/2) Mate pair (MP, 2Kb to 20 Kb) 3/29/2016 38 F F R F R 454/Roche FR Illumina Illumina Slide credit: Aureliano Bombarely BTI Plant Bioinformatics Course 2016
- 39. Implications of Choice of Library 3/29/2016 39 Slide credit: Aureliano Bombarely Consensus sequence (Contig) Reads Scaffold (or Supercontig) Pair Read information NNNNN Pseudomolecule (or ultracontig) F Genetic information (markers) or Optical maps NNNNN NN BTI Plant Bioinformatics Course 2016
- 40. Multiplexing Libraries Use of different tags (4-6 nucleotides) to identify different samples in the same lane/sector. 3/29/2016 40 Slide credit: Aureliano Bombarely AGTCGT TGAGCA AGTCGT AGTCGT AGTCGT AGTCGT TGAGCA TGAGCA TGAGCA TGAGCA AGTCGT AGTCGT AGTCGT AGTCGT TGAGCA TGAGCA TGAGCA TGAGCA Sequencing BTI Plant Bioinformatics Course 2016
- 41. Data!! 3/29/2016 BTI Plant Bioinformatics Course 2016 41
- 42. Fasta files: It is a text-based format for representing either nucleotide sequences or peptide sequences, in which nucleotides or amino acids are represented using single-letter codes. -Wikipedia File Formats 3/29/2016 42 Slide credit: Aureliano Bombarely BTI Plant Bioinformatics Course 2016
- 43. Fastq files: FASTQ format is a text-based format for storing both a biological sequence (usually nucleotide sequence) and its corresponding quality scores. -Wikipedia • Single line ID with at symbol (“@”) in the first column. • Sequences can be in multiple lines after the ID line • Single line with plus symbol (“+”) in the first column to represent the quality line. • Quality ID line may contain ID • Quality values are in multiple lines after the + line but length is identical to sequence 3/29/2016 43 Slide credit: Aureliano Bombarely File Formats BTI Plant Bioinformatics Course 2016
- 44. 3/29/2016 44 Quality control: Encoding Fastq files: !"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33) KLMNOPQRSTUVWXYZ[]^_`abcdefgh Offset by 64 (Phred+64) BTI Plant Bioinformatics Course 2016
- 45. Quality control: Encoding 3/29/2016 45 !"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33) KLMNOPQRSTUVWXYZ[]^_`abcdefgh Offset by 64 (Phred+64) BTI Plant Bioinformatics Course 2016
- 46. 3/29/2016 46 Quality control: Encoding http://en.wikipedia.org/wiki/Phred_quality_score Phred score of a base is: Qphred = -10 log10 (e) where e is the estimated error probability of a base BTI Plant Bioinformatics Course 2016
- 47. Pre-processing: Tools Trimming • FastQC • FASTX toolkit • Trimmomatic • Scythe Joining paired-end reads • fastq-join • FLASH • PANDAseq 3/29/2016 47BTI Plant Bioinformatics Course 2016
- 48. Thank you!! 3/29/2016 BTI Plant Bioinformatics Course 2016 48