1. 10 Billion Piece Jigsaw Puzzles John Cleary Netvalue Ltd. Real Time Genomics

3. million 100 thousand 10 thousand 10 million 100 million billion 10 billion 100 billion thousand hundred

4. Genome Transcriptome Cancer

5. Genomes of … human reference species mouse, chimp, arabidopsis… agricultural species cattle, sheep, pig, … rice, wheat, grape … bacterial disease, human “ecosystem”

6. Differences between … Individuals Populations disease and “quantitative traits” Somatic and tumor genomes Transcriptome of child and parents Bacterial populations of individuals

7. Human Genome 3 billion Nucleotides

8. Shapes of the Jigsaw Pieces CompanyLengths (nt) 45415 - 700 Illumina36 - 150 Complete Genomics36 Ion Torrentupto 200 Oxford Nanopore(?)upto 50,000 Pacific Biosciences100*

9. Differences between genomes - SNPs A C G T T A G T G A A C G T T C G T G A A C G T T G G T G A ~ 1 / 1,000 3,000,000 nt

10. REF: aatgttttctcagaatgtggagaaccttggtgcggacgatgcgcaat_atagggtgggtaccgtccggatac_gctgc______aat______ctgcaatgggaacgacatgatacaatcctgacgggcggtatagaggttctgttgcgtagttagtgttcgtgctgg SIM: T AAGAAT CALL: T G CALL: T T READ: ATGTTTTCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GC READ: TTCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AA READ: TCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG READ: CTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG______A READ: AATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG______AATAAT READ: ATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AA-______GAATAATC READ: ATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG______AATAATC READ: GGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCA READ: GGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCA READ: TGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAAT READ: GAACCTTGGTGCGGACGATGCGCAATTATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAAT READ: AACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGG READ: CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAA READ: CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAA READ: TGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACA READ: TGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATACAAT READ: GCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATC READ: CAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTG READ: _ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGG READ: TAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGG READ: GGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCG READ: TGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTA READ: GGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTAT READ: GTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAG READ: TACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGA READ: CGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGT READ: TTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTT READ: CGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTG READ: TGCAAGAAT______AAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCGTAGT READ: AC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCG READ: AT______AAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCGTAGTTAGTGTT READ: ______AAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCGTAGTTAGTGTTCG

12. Differences between human genomes - MNPs A C G T T A G T G A A C G T T C A G A A C G T T G T G A

13. Differences between human genomes - indels A C G T T A G T G A A C G T T G T G A A C G T T G G T G A ~ 1 / 10,000 300,000

14. Differences between genomes - inserts A C G T T A G T G A Up to 1,000,000 nt total 3,000,000 nt T T A G G A C C C A

15. Differences between genomes – structural variants Tandem Repeat Inversion Copy

16. Solving the Jigsaw Indexing Alignment SNP/MNP/Indel/SV calling Mapping

17. Indexing A C G T T A G T G A A G A C G T T C G T G A A G A C G T T C G T G A A G A C G T T A G T G A A G 4.5 billion

18. Aligning A C G T T A G T G A A G A C G T T C G T G A A G 1.6 billion

19. Cutting Edge Run Human genome (3 billion nt) 1 billion reads of 100 nt coverage of 30 Indexing + Aligning in 27 minutes

20. i7 Quad Core

21. 2 sockets X 4 cores X 2 hyperthreads = 16 48 GB RAM 10 computers 1 TB disk/genome = 500GB + 200GB + 200GB + 0.3GB X thousands of genomes

22. Shapes of the Jigsaw Pieces CompanyLengths (nt) 45415 - 700 Illumina36 - 150 Complete Genomics36 Ion Torrentupto 200 Oxford Nanopore(?)upto 50,000 Pacific Biosciences100*

23. Paired End Reads 100 nt 100 - 1,000 nt Index Align Index Align Match 100 nt

24. Solving the Jigsaw without the picture Indexing Alignment Assembly

25. T A G T G A A G A A T T A C G T T C G T G A A G A C G T T C G T G A A G T A G T G A A G A A T T A C G T T ? G T G A A G A A T T

26. SNP calling 15A13CAC heterozygous SNP 15A4C 5A2C 1A2C Bayesian statistics (SNPs 1/1,000) 31A42C Throw it out

27. REF: aatgttttctcagaatgtggagaaccttggtgcggacgatgcgcaat_atagggtgggtaccgtccggatac_gctgc______aat______ctgcaatgggaacgacatgatacaatcctgacgggcggtatagaggttctgttgcgtagttagtgttcgtgctgg SIM: T AAGAAT CALL: T G CALL: T T READ: ATGTTTTCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GC READ: TTCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AA READ: TCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG READ: CTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG______A READ: AATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG______AATAAT READ: ATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AA-______GAATAATC READ: ATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG______AATAATC READ: GGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCA READ: GGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCA READ: TGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAAT READ: GAACCTTGGTGCGGACGATGCGCAATTATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAAT READ: AACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGG READ: CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAA READ: CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAA READ: TGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACA READ: TGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATACAAT READ: GCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATC READ: CAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTG READ: _ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGG READ: TAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGG READ: GGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCG READ: TGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTA READ: GGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTAT READ: GTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAG READ: TACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGA READ: CGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGT READ: TTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTT READ: CGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTG READ: TGCAAGAAT______AAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCGTAGT READ: AC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCG READ: AT______AAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCGTAGTTAGTGTT READ: ______AAT______CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCGTAGTTAGTGTTCG

28. Comparing twins 3,000,000 SNPs Do any of them differ between the twins? 15A 4C 3A 10C 3G

30. DNA mRNA protein Gene

32. Cancer comparison

33. Copy Number Variants Varying levels of extraction of reads across genome (use differences) Locate boundaries (as accurately as possible) Extract number of variants Use SNPs

36. Metagenomics or what is living on you Mapping reads back onto a database of known bacteria/viruses Many are ambiguous Many don’t map at all Estimate frequency of each species Remove human “contamination”

37. TS1 0.389gi|29611500|ref|NC_004703.1| Bacteroides thetaiotaomicron VPI-5482 plasmid p5482 0.183 gi|187734516|ref|NC_010655.1| Akkermansia muciniphila ATCC BAA-835 0.145gi|150002608|ref|NC_009614.1| Bacteroides vulgatus ATCC 8482 0.037gi|119025018|ref|NC_008618.1| Bifidobacterium adolescentis ATCC 15703 TS4 0.428 gi|29611500|ref|NC_004703.1| Bacteroides thetaiotaomicron VPI-5482 plasmid p5482 0.210 gi|150002608|ref|NC_009614.1| Bacteroides vulgatus ATCC 8482 0.149 gi|60650141|ref|NC_006873.1|Bacteroides fragilis NCTC 9343 plasmid pBF9343 0.037 gi|121999251|ref|NC_008790.1|Campylobacter jejuni subsp. jejuni 81-176 plasmid pTet 0.036 gi|238922432|ref|NC_012781.1|Eubacterium rectale ATCC 33656 TS25 0.752 gi|29611500|ref|NC_004703.1| Bacteroides thetaiotaomicron VPI-5482 plasmid p5482 0.073 gi|150002608|ref|NC_009614.1| Bacteroides vulgatus ATCC 8482 0.041 gi|121999251|ref|NC_008790.1|Campylobacter jejuni subsp. jejuni 81-176 plasmid pTet 0.020 gi|58036264|ref|NC_004307.2|Bifidobacterium longum NCC2705 0.018 gi|189438863|ref|NC_010816.1|Bifidobacterium longum DJO10A

38. Metagenomics Map reads to database Estimate most likely frequencies a hill climbing estimation problem Can anything be done about unmapped reads?