FuzzyPath Assemblies - from Mixed FuzzyPath Assemblies - from Mixed Solexa/454 Datasets to Extremely GC Solexa/454 Datasets to Extremely GC

Biased GenomesBiased Genomes

Zemin NingZemin Ning

The Wellcome Trust Sanger InstituteThe Wellcome Trust Sanger Institute

Assembly StrategyAssembly Strategy

Selexa reads assembler toextend long reads of 1-2Kb

Genome/Chromosome

Capillary reads assemblerPhrap/Phusion

forward-reverse paired reads

30-70 bp

known dist

~500 bp

30-70 bp

Kmer Extension & Repeat JunctionsKmer Extension & Repeat Junctions

Handling of Single Base Variations Handling of Single Base Variations

ACGTAACTACGTAACTAAACAGTTACAGTT00 01 10 11 00 00 01 11 00 01 10 11 00 00 01 11 0000 00 01 00 10 11 11 00 01 00 10 11 11

ACGTAACTACGTAACTCCACAGTTACAGTT00 01 10 11 00 00 01 11 00 01 10 11 00 00 01 11 0101 00 01 00 10 11 11 00 01 00 10 11 11

ACGTAACT ACAGTTACGTAACT ACAGTT00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0101 00 00 00 00 00 00 00 00 00 00 00 00

Fuzzy KmersFuzzy KmersNumber of Mismatches between Two Kmers Number of Mismatches between Two Kmers

Means to handle repeats:Means to handle repeats: - Base quality- Base quality - Read pair- Read pair - Fuzzy kmers- Fuzzy kmers - Closely related reference- Closely related reference - 454 or Sanger reads- 454 or Sanger reads

Kmer Extension & Repeat JunctionsKmer Extension & Repeat Junctions

Pileup of other reads like 454, Sanger etc Pileup of other reads like 454, Sanger etc at a repeat junction at a repeat junction

Consensus

Pileup of Pileup of SolexaSolexa and 454 Reads and 454 Reads

Solexa reads:Number of reads: 3,084,185;Finished genome size: 2,007,491 bp;Read length: 39 and 36 bp;Estimated read coverage: ~55X;Number of 454 reads: 100,000;Read coverage of 454: 10X;

Assembly features: - contig statsTotal number of contigs: 73;Total bases of contigs: 1,999,817 bpN50 contig size: 62,508;Largest contig: 162,190 Averaged contig size: 27,394;Contig coverage over the genome: ~99 %;Contig extension errors: 2Mis-assembly errors: 3

S.SuisS.Suis P1/7 Solexa/454 Assembly P1/7 Solexa/454 Assembly

Solexa reads:Number of reads: 6,000,000;Finished genome size: ~4.8 Mbp;Read length: 2x37 bp;Estimated read coverage: ~92.5 X;Insert size: 170/50-300 bp;

Assembly features: - contig statsSolexa 454

Total number of contigs: 75; 390Total bases of contigs: 4.80 Mbp 4.77 MbN50 contig size: 139,353 25,702Largest contig: 395,600 62,040Averaged contig size: 63,969 12,224Contig coverage on genome: ~99.8 % 99.4%Contig extension errors: 0Mis-assembly errors: 0 4

Salmonella seftenberg Salmonella seftenberg Solexa Solexa Assembly from Pair-End ReadsAssembly from Pair-End Reads

Solexa reads:Number of reads: 7,055,348;Finished genome size: 5.35 Mbp;Read length: 2x36bp;Estimated read coverage: ~95X;Insert size: 170/50-300 bp;

Assembly features: - contig statsTotal number of contigs: 168;Total bases of contigs: 5.19 MbpN50 contig size: 85,886;Largest contig: 337,768 Averaged contig size: 30,886;Contig coverage over the genome: ~99 %;Contig extension errors: 1Mis-assembly errors: 2

E.Coli strain 042 E.Coli strain 042 AssemblyAssembly

Solexa reads:Number of reads: 6,346,317;Finished genome size: 4.7 Mbp;Read length: 33 bp;Estimated read coverage: ~40 X;Shredded reference of SpA: 10X;

Assembly features: - contig statsTotal number of contigs: 66;Total bases of contigs: 4,615,704 bpN50 contig size: 168,793;Largest contig: 401,700 Averaged contig size: 69,934;Contig coverage over the genome: ~98 %;Contig extension errors: 0Mis-assembly errors: 2

Salmonella delhi5 Salmonella delhi5 Solexa AssemblySolexa AssemblyGuided by A Close ReferenceGuided by A Close Reference

The The Malaria Genome Malaria Genome

Project Project

library organism read length Mb sequence genome mean

generated size (Mb) coverage

PCR-free B. pertussis ST24 2 x 76 907 4.1 221

PCR-free E. coli 042 2 x 76 573 5.3 108

PCR-free P. falciparum 3D7 2 x 76 1486 23.0 65

PCR-free B. pertussis ST24 2 x 36 452 4.1 110

PCR-free P. falciparum 3D7 2 x 36 1008 23.0 44

PCR-free E. coli 042 2 x 36 958 5.3 181

standard-245 P. falciparum 3D7 2 x 35 2198 23.0 96

standard-368 P. falciparum 3D7 2 x 35 2628 23.0 115

standard-851 P. falciparum 3D7 2 x 35 474 23.0 21

standard-883 P. falciparum clin 2 x 36 3994 23.0 175

Datasets with Various GC ContentDatasets with Various GC Content

GC

68.0%

50.5%

19.0%

19.0%

50.8%

19.0%

68.0%

19.0%

19.0%

19.0%

0

1

2

3

4

5

6

7

8

9

10

11

12

0 10 20 30 40 50 60 70 80 90 100Depth of genome base coverage

Frac

tion

of u

nmak

ed g

enom

e (%

)

3D7:PCR-free-36bp-43x3D7:PCR-free-76bp-64xPFClin:run883-36bp-50xPFClin:run883-36bp-170x3D7:run368-35bp-114x3D7:run245-35bp-95x3D7:run851-35bp-21x

0

10

20

30

40

50

60

70

80

90

100

1 10 100 1000Depth of genome base coverage

Acc

umul

ated

frac

tion

of u

nmak

sed

geno

me

(%)

3D7:PCR-free-36bp-43x

3D7:PCR-free-76bp-64x

PFClin:run883-36bp-170x

3D7:run368-35bp-114x

3D7:run245-35bp-95x

3D7:run851-35bp-21x

0

0.1

0.2

0.3

0.4

0.5

0.6

0 20 40 60 80 100 120 140 160 180 200Depth of genome base coverage

GC

con

tent

(%)

3D7:PCR-free-36bp-43x

3D7:PCR-free-76bp-64x

PFClin:run883-36bp-170x

3D7:run368-35bp-114x

3D7:run245-35bp-95x

3D7:run851-35bp-21x

0

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8 10 12 14 16 18 20

Depth of duplication

Per

cent

age

of m

atch

ed r

easd

s (%

) 3D7:PCR-free-36bp-43x

3D7:PCR-free-76bp-64x

PFClin:run883-36bp-170x

3D7:run368-35bp-114x

3D7:run245-35bp-95x

3D7:run851-35bp-21x

Solexa reads: 2x36 bp 2x76 bpNumber of reads: 14.0m 9.77mFinished genome size: 23 Mbp 23 MbpEstimated read coverage: 43x 64xInsert size: 170 bp 170 bp

Assembly features:Total number of contigs: 26,926 22839Total bases of contigs: 19.2 Mbp 21.1 MbN50 contig size: 1456 1621Largest contig: 9106 9825Averaged contig size: 706 923Contig coverage on genome: ~83.5 % 91.7%Contig extension errors: ? ?Mis-assembly errors: ? ?

Malaria 3D7 AssembliesMalaria 3D7 Assemblies

Acknowledgements:

Yong Gu Ben Blackburne Hannes Ponstingl Daniel Turner Michael Quail Tony Cox Richard Durbin