Sequencing
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Surya Saha Sol Genomics Network (SGN) Boyce Thompson Institute, Ithaca, NY @SahaSurya BTI PGRP Summer Internship Program 2014
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Sequencing
- 1. Surya Saha Sol Genomics Network (SGN) Boyce Thompson Institute, Ithaca, NY ss2489@cornell.edu // @SahaSurya BTI PGRP Summer Internship Program 2014 http://www.acgt.me/blog/2014/3/7/next-generation-sequencing-must-die
- 2. Why Sequencing? • Targeted interrogation of genome • Economical • Technological developments • High-throughput assays • But requires subsequent validation 7/8/2014 BTI PGRP Summer Internship Program 2014 2
- 3. 1953 DNA Structure discovery 1977 2012 Sanger DNA sequencing by chain-terminating inhibitors 1984 Epstein-Barr virus (170 Kb) 1987Abi370 Sequencer 1995 2001 Homo sapiens (3.0 Gb) 2005 454 Solexa Solid 2007 2011 Ion Torrent PacBio Haemophilus influenzae (1.83 Mb) 2013 Slide credit: Aureliano Bombarely Sequencing over the Ages Illumina Illumina Hiseq X 454 7/8/2014 BTI PGRP Summer Internship Program 2014 3 Pinus taeda (24 Gb)
- 4. First generation sequencing 7/8/2014 BTI PGRP Summer Internship Program 2014 4
- 5. Sanger method 7/8/2014 BTI PGRP Summer Internship Program 2014 5 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
- 6. Sanger method 7/8/2014 BTI PGRP Summer Internship Program 2014 6 http://bit.ly/1g6Cudq http://bit.ly/1lcQO4J
- 7. First generation sequencing • Very high quality sequences (99.999%) • Very low throughput 7/8/2014 BTI PGRP Summer Internship Program 2014 7 Run Time Read Length Reads / Run Total nucleotides sequenced Cost / MB Capillary Sequencing (ABI3730xl) 20m-3h 400-900 bp 96 or 386 1.9-84 Kb $2400 http://bit.ly/1clLps3 http://1.usa.gov/1cLqIRd
- 8. Use the specific technology used to generate the data – Illumina Hiseq/Miseq/NextSeq – Pacific Biosciences RS1/RSII – Ion Torrent Proton/PGM – SOLiD 7/8/2014 BTI PGRP Summer Internship Program 2014 8 http://www.acgt.me/blog/2014/3/10/next-generation- sequencing-must-diepart-2
- 9. 454 Pyrosequencing One purified DNA fragment, to one bead, to one read. 7/8/2014 BTI PGRP Summer Internship Program 2014 9 http://bit.ly/1ehwxWN GS FLX Titanium http://bit.ly/1ehAcEh
- 10. Illumina 7/8/2014 BTI PGRP Summer Internship Program 2014 10 Output 15 Gb 120 GB 1000 GB 1800 GB Number of Reads 25 Million 400 Million 4 Billion 6 Billion Read Length 2x300 bp 2x150 bp 2x125 bp (2x250 update mid-2014) 2x150 bp Cost $99K $250K $740K $10M Source: Illumina
- 11. Illumina 7/8/2014 BTI PGRP Summer Internship Program 2014 11 http://1.usa.gov/1fP9ybl
- 12. Illumina:Moleculo 7/8/2014 BTI PGRP Summer Internship Program 2014 12 http://bit.ly/1aEPOBn
- 13. Pacific Biosciences SMRT sequencing Single Molecule Real Time sequencing 7/8/2014 BTI PGRP Summer Internship Program 2014 13 http://bit.ly/1naxgTe
- 14. Pacific Biosciences SMRT sequencing Error correction methods 7/8/2014 BTI PGRP Summer Internship Program 2014 14 Hierarchical genome-assembly process (HGAP) PBJelly Enlish et al., PLOS One. 2012 PBJelly
- 15. 7/8/2014 Centre for Agricultural Bioinformatics, Pusa 15 Pacific Biosciences SMRT sequencing Read Lengths
- 16. Oxford Nanopore 7/8/2014 Centre for Agricultural Bioinformatics, Pusa 16 https://www.nanoporetech.com/ • No data yet?? • Error model http://erlichya.tumblr.com/post/66376172948/hands-on- experience-with-oxford-nanopore-minion
- 17. Others • Ion Torrent Proton/PGM • Helicos • Nabsys • SOLiD • …… 7/8/2014 BTI PGRP Summer Internship Program 2014 17
- 18. Comparison 7/8/2014 BTI PGRP Summer Internship Program 2014 18
- 19. Next generation sequencing 7/8/2014 BTI PGRP Summer Internship Program 2014 19 Run Time Read Length Quality Total nucleotides sequenced Cost /MB 454 Pyrosequencing 24h 700 bp Q20-Q30 0.7 GB $10 Illumina Miseq 27h 2x250bp > Q30 15 GB $0.15 Illumina Hiseq 2500 11days 2x125bp >Q30 1000 GB $0.05 Ion torrent 2h 400bp >Q20 50MB-1GB $1 Pacific Biosciences 2h 8.5-20kb >Q30 consensus >Q10 single 400-850MB /SMRT cell $0.33-$1 http://bit.ly/1clLps3 http://1.usa.gov/1cLqIRd
- 20. http://omicsmaps.com/ Next Generation Genomics: World Map of High-throughput Sequencers BTI PGRP Summer Internship Program 20147/8/2014 20
- 21. 7/8/2014 BTI PGRP Summer Internship Program 2014 21
- 22. Real cost of Sequencing!! Sboner, Genome Biology, 2011 7/8/2014 22Centre for Agricultural Bioinformatics, Pusa
- 23. Library Types Single end Pair end (PE, 150-800 bp, Fwd:/1, Rev:/2) Mate pair (MP, 2Kb to 20 Kb) 7/8/2014 BTI PGRP Summer Internship Program 2014 23 F F R F R 454/Roche FR Illumina Illumina Slide credit: Aureliano Bombarely
- 24. Implications of Choice of Library 7/8/2014 BTI PGRP Summer Internship Program 2014 24 Slide credit: Aureliano Bombarely Consensus sequence (Contig) Reads Scaffold (or Supercontig) Pair Read information NNNNN Pseudomolecule (or ultracontig) F Genetic information (markers) NNNNN NN
- 25. Multiplexing Libraries Use of different tags (4-6 nucleotides) to identify different samples in the same lane/sector. 7/8/2014 BTI PGRP Summer Internship Program 2014 25 Slide credit: Aureliano Bombarely AGTCGT TGAGCA AGTCGT AGTCGT AGTCGT AGTCGT TGAGCA TGAGCA TGAGCA TGAGCA AGTCGT AGTCGT AGTCGT AGTCGT TGAGCA TGAGCA TGAGCA TGAGCA Sequencing
- 26. 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 7/8/2014 BTI PGRP Summer Internship Program 2014 26 Slide credit: Aureliano Bombarely
- 27. 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 should be identical to sequence 7/8/2014 BTI PGRP Summer Internship Program 2014 27 Slide credit: Aureliano Bombarely File Formats
- 28. 7/8/2014 BTI PGRP Summer Internship Program 2014 28 Quality control: Encoding Fastq files: !"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33) KLMNOPQRSTUVWXYZ[]^_`abcdefgh Offset by 64 (Phred+64)
- 29. Quality control: Encoding 7/8/2014 BTI PGRP Summer Internship Program 2014 29 !"#$%&'()*+,-./0123456789 Offset by 33 (Phred+33) KLMNOPQRSTUVWXYZ[]^_`abcdefgh Offset by 64 (Phred+64)
- 30. 7/8/2014 BTI PGRP Summer Internship Program 2014 30 Quality control: Encoding http://bit.ly/N28yUd Phred score of a base is: Qphred = -10 log10 (e) where e is the estimated probability of a base being wrong
- 31. Pre-processing: Tools Trimming • FastQC • FASTX toolkit • Trimmomatic • Scythe Joining paired-end reads • fastq-join • FLASH • PANDAseq 7/8/2014 BTI PGRP Summer Internship Program 2014 31
- 32. Pre-processing: Error correction 7/8/2014 BTI PGRP Summer Internship Program 2014 32
- 33. Thank you!! 7/8/2014 BTI PGRP Summer Internship Program 2014 33