Genomics I: High throughput sequencing

1. Genomics I: High throughput sequencing Jim Noonan Department of Genetics

2. A working definition of genomics • The global study of how biological information is encoded in • genome sequence • Genes • Regulatory sequences • Genetic variation • How this information is read out to produce distinct biological • outcomes • Gene expression and regulation • Cellular identity, differentiation and development • Phenotypic variation among individuals and species

3. Genomes are vast information repositories Human 3 Gb • kb = 1000 bp • Mb = 1x106bp • Gb = 1x109bp • Tb = 1x1012bp • Pb = 1x1015bp 1 Gb 10 Gb 100 Gb

4. Reference genomes

5. Genome assembly and annotation >>109 sequencing reads 36 bp - 1 kb 3 Gb

6. Genome assembly Find overlapping reads Join contigs into scaffolds mate pair scaffold Join scaffolds into “finished”sequence anchored on chromosomes AGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAG Assembly quality criteria: Accuracy: number of errors (Human << 1/100,000 bp) Contiguity: number of gaps (Human: est. 357) Generate reads Coverage: Average number of reads representing a particular position in the assembly Human, Mouse, Rat: > 20x Chimpanzee:~6x Squirrel: ~2x Assemble reads into contigs contig Scaffold_0: 12,865,123 – 12,965-110 Chr5: 133,876,119 – 134,876,119

7. ATATCATGCTTGTCATTCGTTTATCAGAGGCCAAATGTTTTTCTTTGTAAACGTGTGTAAAACATTCTCAGAATTTTAAACAATAACAAATCAGGGCTGAATGTGGCCAACATGCAAAGAGGAAATCTCCCATCTGTCCAAATCAAACAGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAGAGAACATGCCAAAAGTTTAAGCAAGAAGAAAACAAAGACTGTTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCAACATGCAAAGAGGAAATCTCCATATCATGCTTGTCATTCGTTTATCAGAGGCCAAATGTTTTTCTTTGTAAACGTGTGTAAAACATTCTCAGAATTTTAAACAATAACAAATCAGGGCTGAATGTGGCCAACATGCAAAGAGGAAATCTCCCATCTGTCCAAATCAAACAGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAGAGAACATGCCAAAAGTTTAAGCAAGAAGAAAACAAAGACTGTTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCAACATGCAAAGAGGAAATCTCCATATCATGCTTGTCATTCGTTTATCAGAGGCCAAATGTTTTTCTTTGTAAACGTGTGTAAAACATTCTCAGAATTTTAAACAATAACAAATCAGGGCTGAATGTGGCCAACATGCAAAGAGGAAATCTCCCATCTGTCCAAATCAAACAGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAGAGAACATGCCAAAAGTTTAAGCAAGAAGAAAACAAAGACTGTTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCAACATGCAAAGAGGAAATCTCCATATCATGCTTGTCATTCGTTTATCAGAGGCCAAATGTTTTTCTTTGTAAACGTGTGTAAAACATTCTCAGAATTTTAAACAATAACAAATCAGGGCTGAATGTGGCCAACATGCAAAGAGGAAATCTCCCATCTGTCCAAATCAAACAGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAGAGAACATGCCAAAAGTTTAAGCAAGAAGAAAACAAAGACTGTTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCAACATGCAAAGAGGAAATCTCCATATCATGCTTGTCATTCGTTTATCAGAGGCCAAATGTTTTTCTTTGTAAACGTGTGTAAAACATTCTCAGAATTTTAAACAATAACAAATCAGGGCTGAATGTGGCCAACATGCAAAGAGGAAATCTCCCATCTGTCCAAATCAAACAGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAGAGAACATGCCAAAAGTTTAAGCAAGAAGAAAACAAAGACTGTTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCAACATGCAAAGAGGAAATCTCCATATCATGCTTGTCATTCGTTTATCAGAGGCCAAATGTTTTTCTTTGTAAACGTGTGTAAAACATTCTCAGAATTTTAAACAATAACAAATCAGGGCTGAATGTGGCCAACATGCAAAGAGGAAATCTCCCATCTGTCCAAATCAAACAGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAGAGAACATGCCAAAAGTTTAAGCAAGAAGAAAACAAAGACTGTTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCAACATGCAAAGAGGAAATCTCCATATCATGCTTGTCATTCGTTTATCAGAGGCCAAATGTTTTTCTTTGTAAACGTGTGTAAAACATTCTCAGAATTTTAAACAATAACAAATCAGGGCTGAATGTGGCCAACATGCAAAGAGGAAATCTCCCATCTGTCCAAATCAAACAGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAGAGAACATGCCAAAAGTTTAAGCAAGAAGAAAACAAAGACTGTTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCAACATGCAAAGAGGAAATCTCCATATCATGCTTGTCATTCGTTTATCAGAGGCCAAATGTTTTTCTTTGTAAACGTGTGTAAAACATTCTCAGAATTTTAAACAATAACAAATCAGGGCTGAATGTGGCCAACATGCAAAGAGGAAATCTCCCATCTGTCCAAATCAAACAGTTGTATTATTAGAAACTGAGGGCTAAAAACTGTGCACATACACAGACACACATATTATTTTAATATAGATTTTCAATAATTGGTCTAGGATAAGGATAATATACAGAGAACATGCCAAAAGTTTAAGCAAGAAGAAAACAAAGACTGTTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCAACATGCAAAGAGGAAATCTCC

8. Genome annotation • Genes: • Coding, noncoding, miRNA, etc. • Isoforms • Expression ~3 billion bp ACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTAGAAAGACAATGAAACAGAGCCATGTGACCAATGAGAGAGATGAGGGTGGCAGCAGCCTGTTTTAGATAAGGTACCTGATTGGTGGGATTGGAAGACCTCTCTGAGATTAGTGTCTTCAGATATGCCTTAATGATATGAAAGAACCATTCATGGGAAGGCCTAGCATTAAAAACCGTCTAGGCAGAATGAGCAGCAAGTGCAAGGGTCCTGGATAGGAATGAGCTGGATATACTCAAGGAAGAAAGAGAAACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACTAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTACTATGGAAAAATGAAAATAGATTTTAAAACATGTTAATTCACGTTACTTTTTGTTAAATTTACTTTTCTTCTTTCACTTCTTACCTGTCAATGTTATTAATATTTTTAGGAACAATAAATCACATTAATTCCTTATCTCATGTGAAATTTCATATTTATGATTGATACCTTTAAATGTCATTTGTTGAAGGAAGATTATTCATTTTTTCATTCAATAAATATTTTTTAGAATAATAAGTCCCAGGCACAAGACCAGTATTATGTTCTAGGCATTGGGGATACCATGTTCACAAGACAGACTATGATTTACAGGATCAGATGTGGACTCTCAAATTCGACTGAGAATAAAACAGACACAAACAAGTAAATAAAGTTAATTTCAAGTTGTAATTGATGCTATCCCAGGCACAAGACCA…. • Genetic variation: • SNPs and CNVs Sequence conservation • Regulatory sequences: • Promoters • Enhancers • Insulators • Epigenetics: • DNA methylation • Chromatin

9. Portals to access and interpret genomes UCSC Genome Browser(genome.ucsc.edu): Visualization, data recovery, simple analysis (also http://genome-preview.ucsc.edu/) ENSEMBL (ensembl.org): Visualization, data recovery, simple analysis Integrative Genomics Viewer (broadinstitute.orgsoftware/igv/): Local genome viewer (visualize local and remote data) Galaxy (main.g2.bx.psu.edu): Complex data analysis and workflows

10. Annotation depth varies by species • Human, Mouse (Fly, Worm, Yeast): • Chromosome assemblies • Dense gene and regulatory maps, variation, etc. • Other models (Dog, Chicken, Zebrafish): • Chromosome assemblies • Partial gene maps; variation; little regulatory data • Low coverage vertebrate genomes: • Scaffold assemblies • Few annotated genes • Used for comparative purposes

11. Density of biological information in the human genome Chr5: 133,876,119 – 134,876,119 Genes Transcription Histone mods TF binding Mouse orthology SNPs Repeats

12. Determining the biological state of cells, tissues and organisms requires the quantification of sequence information • Gene expression • Protein-DNA interactions (ChIP) • DNA-DNA interactions (3C/4C/5C) • Chromatin state • DNA methylation • Genetic variation (SNPs/CNVs) Indirect measures Direct measures CTATGATCAGTC... TCAATCTGATCTG... GGACTTCGAGATC... AAGTCGCTGACGT... microarrays, PCR, etc. Sequencing Sequence as the readout for biological processes

13. First-generation sequencing technology • Sanger sequencing • Parallelization in human genome project Current massively parallel sequencing strategies • “SecondGeneration” • 454 • Illumina • SOLiD • Ion Torrent & Ion Proton • Sequencing Services (Complete Genomics) • “Third Generation” • Pacific Biosciences • Oxford Nanopore Outline

14. Throughput • Number of high quality bases per unit time • Number of independent samples run in parallel - multiplexing • Difficulty of sample prep Yield • Number of useful/mappable reads per sample • Read length Cost • Per run and per base • Equipment • Reagents • Labor • Analysis Metrics for evaluating sequencing methods The goal is to increase throughput and yield while reducing cost

15. 1980 Nobel Prize in chemistry gels read by hand phi X 174 ~5300 bp • radiolabeled dideoxyNTPs • one lane per nucleotide • 800 bp reads • low throughput (several kb/gel) Sanger sequencing (1975-1977)

16. Industrialization of Sanger sequencing, • library construction, sample preparation, • analysis, etc. • $3 billion total cost • 1 Gb/month at largest centers (2005) • YCGA = 9.6 Tb per month (2011) Sequencing the reference human genome (1990-present; ‘finished’ 2003)

17. “Democratizing” sequencing production • Massive parallelization • Reduction in per-base cost • Eliminate need for huge infrastructure • Millions of reads - >1Gb sequence per run Novel sequencing applications • RNA-seq Countingapplications • ChIP-seq • Methyl-seq • Whole-genome and targeted resequencing Challenges • Read length • Quality • Data analysis and storage Second-generation sequencing

18. Short read technologies: Illumina Flow cell

19. Flow cells A flow cell is a thick glass slide with 8 channels or lanes Each lane is randomly coated with a lawn of oligos that are complementary to library adapters P5 oligo P7 oligo Adapter1 Adapter2 Insert Sequencing Primer

20. Illumina sequencing Cluster PCR on flow cell (8 lanes) Attach to flow cell Cluster generation ‘bridge PCR’ Reverse termination Add next base Add base Scan flow cell Sequencing by synthesis with reversible dye terminators 1 cycle

23. HiSeq 2500 1 Instrument – 2 Run Modes High Output Mode 600 Gb in ~10.5 days Current v3 flow cell Current v3 reagents cBot required Rapid Run Mode 120Gb in ~1 day New 2-lane flow cell New reagents No cBot required User configurable 6 human genomes in 10.5 days 1 human genome in a day Highest Output Fastest turnaround

24. MiSeq • Run-times • 50 cycle – 4 hours • 300 cycle – 27 hours • Two sequencing options • 50 cycles • 300 cycles (2x150 bp) • One lane • 6-7 million clusters • Up to 8 billion bases (300 cycles) Ideal for: R&D, CLIA, small genomes and projects where longer reads are important

25. Ion Torrent and Ion Proton • Sequencing on • semiconductor chip

26. Ion Torrent sequencing chemistry When a nucleotide is incorporated into a strand of DNA, a hydrogen ion is released as a byproduct. The H ion carries a charge which the PGM’s ion sensor can detect as a base.

27. Ion Torrent yields (v1; v2 is higher) • Ion PI chip: >165 million wells per chip: 8 to 10 Gb data per run • Ion PII chips: ~100 Gb of data in ~4 hours

28. Advantages and limitations Advantages • Low equipment cost • Rapid run times: 3 to 4 hours • Simple Chemistry Limitations • Homopolymers detection • Error rates • Slow on introducing newer chips: Overpromise • PGM and Proton: two separate systems • Library prep: Emulsion PCR

29. Third-generation sequencing High-throughput single molecule sequencing in real time at low cost Pacific Biosciences • Sequence in real time with fluorescent NTPs • Rate limited by processivity of polymerase • Very long reads possible (6 kb) • Not well parallelized (few reads)

30. Sequencing in real time: Pacific Biosciences SMRT cells Zero Mode Waveguides

31. PacBiosequencing strategy

32. PacBiosequencing strategy

33. Applications • Targeted sequencing • SNP and structure variants detection • Repetitive regions • Full length transcript profiling • De novo assembly and genome finishing • Bacterial genomes • Fungal genomes • Gap-captured sequencing • Targeted captured sequencing • Base modifications detection • Methylation • DNA damage YCGA PacBio RS **Projects at YCGA Shrikant Mane

34. PacBiovsIllumina Shrikant Mane

35. Oxford Nanopore Exonuclease Cyclodextrin Lipid bilayer

36. Oxford Nanopore GridION: enables scaled-up measurements of multiple nanopores and analysis of data in real time MiniION

37. Advantages and limitations • Nanopores offer a label-free, electrical, single-molecule • DNA sequencing method • No costly fluorescent labeling reagents • No need for expensive optical hardware and sophisticated • instrumentation to detect DNA bases • Runs as long as needed • High error rates ~5% • Not available yet

38. Conclusions • High-throughput sequencing has become democratized - • moved out of industrial-scale genome centers • Sequence is no longer limiting - next generation of sequencers will • make sequencing very inexpensive • Earlier methods for counting / resequencing applications are largely obsolete • Scale of data production outstripping our ability to store and analyze it • Next: Applications of the technology