10 Billion Piece Jigsaw Puzzles

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10 Billion Piece Jigsaw Puzzles

• John Cleary
• Netvalue Ltd.
• Real Time Genomics

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100 billion
10 billion
billion

• 100 million

10 million
million
100 thousand
thousand
10 thousand
hundred
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Genome Transcriptome Cancer
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Genomes of

• human
• reference speciesmouse, chimp, arabidopsis
• agricultural speciescattle, sheep, pig, rice,
  wheat, grape
• bacterialdisease, human ecosystem

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Differences between

• Individuals
• Populations disease and quantitative traits
• Somatic and tumor genomes
• Transcriptome of child and parents
• Bacterial populations of individuals

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Human Genome
3 billion Nucleotides
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Shapes of the Jigsaw Pieces
Company Lengths (nt)
454 15 - 700
Illumina 36 - 150
Complete Genomics 36
Ion Torrent upto 200
Oxford Nanopore(?) upto 50,000
Pacific Biosciences 100
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Differences betweengenomes - SNPs
1 / 1,000 3,000,000 nt
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REF aatgttttctcagaatgtggagaaccttggtgcggacgatgcgca
at_atagggtgggtaccgtccggatac_gctgc______aat______ct
gcaatgggaacgacatgatacaatcctgacgggcggtatagaggttctgt
tgcgtagttagtgttcgtgctgg SIM
T
AAGAAT SIM T

AAGAAT CALL T

G CALL T

T READ ATGTTTTCTCAGAATGTGGTGAACCTTGGTGCGGACGATGC
GCAAT_ATAGGGTGGGTACCGTCCGGATAC_GC

READ
ATGTTTTCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATA
GGGTGGGTACCGTCCGGATAC_GC

READ TTCTCAGAATGTGGTGAAC
CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GC
TGC______AA

READ TCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCG
CAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG

READ
CTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGG
TACCGTCCGGATAC_GCTGC______AAG______A

READ AATGTGGTGAACCTTGGTG
CGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC____
__AAG______AATAAT
READ
ATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTG
GGTACCGTCCGGATAC_GCTGC______AA-______GAATAATC

READ
ATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGT
CCGGATAC_GCTGC______AAG______AATAATC

READ GGTGAACCTTGGTGCGGACG
ATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT_
_____CTGCA
READ
GGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACC
GTCCGGATAC_GCTGC______AAGAATAATCTGCA

READ
GGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGG
ATAC_GCTGC______AAGAATAATCTGCA

READ TGAACCTTGGTGCGGACGATGC
GCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT_____
_CTGCAAT
READ
GAACCTTGGTGCGGACGATGCGCAATTATAGGGTGGGTACCGTCC
GGATAC_GCTGC______AAGAATAATCTGCAAT

READ
AACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC
_GCTGC______AAGAATAATCTGCAATGG

READ AACCTTGGTGCGGACGATGCG
CAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAAT
CTGCAATGG
READ
CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCG
GATAC_GCTGCAAGAATAAT______CTGCAATGGGAA

READ
CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GC
TGC______AAGAATAATCTGCAATGGGAA

READ
TGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAA
GAATAAT______CTGCAATGGGAACGACA
READ

TGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT__
____CTGCAATGGGAACGACATGATACAAT
READ
GCGCAAT_ATAGGGTGGGTACCG
TCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGA
TACAATC
READ
CAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG
AATAATCTGCAATGGGAACGACATGATACAATCCTG
READ

_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGC
AATGGGAACGACATGATACAATCCTGACGG
READ
TAGGGTGGGTACCGTCCGGATA
C_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCC
TGACGGG
READ
GGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______
CTGCAATGGGAACGACATGATACAATCCTGACGGGCG
READ

TGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGA
ACGACATGATACAATCCTGACGGGCGGTA
READ
GGGTACCGTCCGGATAC_GCTGC
______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGG
CGGTAT READ

GTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGG
GAACGACATGATACAATCCTGACGGGCGGTATAG
READ

TACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGA
CATGATACAATCCTGACGGGCGGTATAGA
READ
CGTCCGGATAC_GCTGC____
__AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGT
ATAGAGGT READ

TTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAAC
GACATGATACAATCCTGACGGGCGGTATAGAGGTT
READ

CGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATA
CAATCCTGACGGGCGGTATAGAGGTTCTG
READ
TGCAAGAAT______AAT__
____CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGG
TTCTGTTGCGTAGT READ

AC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGA
TACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCG
READ

AT______AAT______CTGCAATGGGAACGACATGATACAATCCTGACG
GGCGGTATAGAGGTTCTGTTGCGTAGTTAGTGTT READ

______AAT______CTGCAAT
GGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCGT
AGTTAGTGTTCG
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Differences between humangenomes - MNPs
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Differences between humangenomes - indels
1 / 10,000 300,000
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Differences between genomes - inserts
T T A G G A C C C A
Up to 1,000,000 nt total 3,000,000 nt
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Differences between genomes structural variants
Tandem Repeat
Inversion
Copy
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Solving the Jigsaw

• Indexing
• Alignment
• SNP/MNP/Indel/SV calling

Mapping
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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 A G T G A A G
A C G T T C G T G A A G
4.5 billion
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Aligning
1.6 billion
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Cutting Edge Run

• Human genome (3 billion nt)
• 1 billion reads of 100 nt coverage of 30
• Indexing Aligning in 27 minutes

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i7 Quad Core
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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
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Shapes of the Jigsaw Pieces
Company Lengths (nt)
454 15 - 700
Illumina 36 - 150
Complete Genomics 36
Ion Torrent upto 200
Oxford Nanopore(?) upto 50,000
Pacific Biosciences 100
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Paired End Reads
100 nt
100 - 1,000 nt
Index Align
Index Align
Match
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Solving the Jigsawwithout the picture

• Indexing
• Alignment

Assembly
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Assembly
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 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
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SNP calling
15A 13C
AC heterozygous SNP
Throw it out
31A 42C
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REF aatgttttctcagaatgtggagaaccttggtgcggacgatgcgca
at_atagggtgggtaccgtccggatac_gctgc______aat______ct
gcaatgggaacgacatgatacaatcctgacgggcggtatagaggttctgt
tgcgtagttagtgttcgtgctgg SIM
T
AAGAAT SIM T

AAGAAT CALL T

G CALL T

T READ ATGTTTTCTCAGAATGTGGTGAACCTTGGTGCGGACGATGC
GCAAT_ATAGGGTGGGTACCGTCCGGATAC_GC

READ
ATGTTTTCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATA
GGGTGGGTACCGTCCGGATAC_GC

READ TTCTCAGAATGTGGTGAAC
CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GC
TGC______AA

READ TCTCAGAATGTGGTGAACCTTGGTGCGGACGATGCG
CAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG

READ
CTCAGAATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGG
TACCGTCCGGATAC_GCTGC______AAG______A

READ AATGTGGTGAACCTTGGTG
CGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC____
__AAG______AATAAT
READ
ATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTG
GGTACCGTCCGGATAC_GCTGC______AA-______GAATAATC

READ
ATGTGGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGT
CCGGATAC_GCTGC______AAG______AATAATC

READ GGTGAACCTTGGTGCGGACG
ATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT_
_____CTGCA
READ
GGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACC
GTCCGGATAC_GCTGC______AAGAATAATCTGCA

READ
GGTGAACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGG
ATAC_GCTGC______AAGAATAATCTGCA

READ TGAACCTTGGTGCGGACGATGC
GCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT_____
_CTGCAAT
READ
GAACCTTGGTGCGGACGATGCGCAATTATAGGGTGGGTACCGTCC
GGATAC_GCTGC______AAGAATAATCTGCAAT

READ
AACCTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC
_GCTGC______AAGAATAATCTGCAATGG

READ AACCTTGGTGCGGACGATGCG
CAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAAT
CTGCAATGG
READ
CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCG
GATAC_GCTGCAAGAATAAT______CTGCAATGGGAA

READ
CTTGGTGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GC
TGC______AAGAATAATCTGCAATGGGAA

READ
TGCGGACGATGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAA
GAATAAT______CTGCAATGGGAACGACA
READ

TGCGCAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT__
____CTGCAATGGGAACGACATGATACAAT
READ
GCGCAAT_ATAGGGTGGGTACCG
TCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGA
TACAATC
READ
CAAT_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAG
AATAATCTGCAATGGGAACGACATGATACAATCCTG
READ

_ATAGGGTGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGC
AATGGGAACGACATGATACAATCCTGACGG
READ
TAGGGTGGGTACCGTCCGGATA
C_GCTGC______AAGAATAATCTGCAATGGGAACGACATGATACAATCC
TGACGGG
READ
GGGTGGGTACCGTCCGGATAC_GCTGCAAGAATAAT______
CTGCAATGGGAACGACATGATACAATCCTGACGGGCG
READ

TGGGTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGA
ACGACATGATACAATCCTGACGGGCGGTA
READ
GGGTACCGTCCGGATAC_GCTGC
______AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGG
CGGTAT READ

GTACCGTCCGGATAC_GCTGC______AAGAATAATCTGCAATGG
GAACGACATGATACAATCCTGACGGGCGGTATAG
READ

TACCGTCCGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGA
CATGATACAATCCTGACGGGCGGTATAGA
READ
CGTCCGGATAC_GCTGC____
__AAGAATAATCTGCAATGGGAACGACATGATACAATCCTGACGGGCGGT
ATAGAGGT READ

TTCCGGATAC_GCTGC______AAGAATAATCTGCAATGGGAAC
GACATGATACAATCCTGACGGGCGGTATAGAGGTT
READ

CGGATAC_GCTGCAAGAATAAT______CTGCAATGGGAACGACATGATA
CAATCCTGACGGGCGGTATAGAGGTTCTG
READ
TGCAAGAAT______AAT__
____CTGCAATGGGAACGACATGATACAATCCTGACGGGCGGTATAGAGG
TTCTGTTGCGTAGT READ

AC_GCTGC______AAGAATAATCTGCAATGGGAACGACATGA
TACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCG
READ

AT______AAT______CTGCAATGGGAACGACATGATACAATCCTGACG
GGCGGTATAGAGGTTCTGTTGCGTAGTTAGTGTT READ

______AAT______CTGCAAT
GGGAACGACATGATACAATCCTGACGGGCGGTATAGAGGTTCTGTTGCGT
AGTTAGTGTTCG
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Comparing twins
3,000,000 SNPs Do any of them differ between the
twins?
15A 4C
3A 10C 3G
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Gene
DNA
mRNA
protein
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Cancer comparison
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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

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Metagenomics or what is living on you

• Mapping reads back onto a database of known
  bacteria/viruses
• Many are ambiguous
• Many dont map at all
• Estimate frequency of each species
• Remove human contamination

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TS1 0.389 gi29611500refNC_004703.1
Bacteroides thetaiotaomicron VPI-5482 plasmid
p5482 0.183 gi187734516refNC_010655.1
Akkermansia muciniphila ATCC BAA-835 0.145 gi150
002608refNC_009614.1 Bacteroides vulgatus
ATCC 8482 0.037 gi119025018refNC_008618.1
Bifidobacterium adolescentis ATCC 15703 TS4
0.428 gi29611500refNC_004703.1 Bacteroides
thetaiotaomicron VPI-5482 plasmid p5482 0.210
gi150002608refNC_009614.1 Bacteroides
vulgatus ATCC 8482 0.149 gi60650141refNC_0068
73.1 Bacteroides fragilis NCTC 9343 plasmid
pBF9343 0.037 gi121999251refNC_008790.1 Camp
ylobacter jejuni subsp. jejuni 81-176 plasmid
pTet 0.036 gi238922432refNC_012781.1 Eubacte
rium rectale ATCC 33656 TS25 0.752
gi29611500refNC_004703.1 Bacteroides
thetaiotaomicron VPI-5482 plasmid p5482 0.073
gi150002608refNC_009614.1 Bacteroides
vulgatus ATCC 8482 0.041 gi121999251refNC_008
790.1 Campylobacter jejuni subsp. jejuni 81-176
plasmid pTet 0.020 gi58036264refNC_004307.2
Bifidobacterium longum NCC2705 0.018
gi189438863refNC_010816.1 Bifidobacterium
longum DJO10A
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Metagenomics

• Map reads to database
• Estimate most likely frequenciesa hill climbing
  estimation problem
• Can anything be done about unmapped reads?

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