Evaluation of the impact of error correction algorithms on SNP calling.

Single Nucleo,de Polymorphism (SNP) valida,on for food-‐borne pathogen

strain level iden,fica,on Nathan Olson

Na,onal Ins,tute of Standards and Technology

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To promote U.S. innova#on and industrial compe##veness by advancing measurement science, standards, and technology in ways that enhance economic security and improve our quality of life.

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© Rob

ert R

athe

NIST

NIST and Microbial Iden,fica,on Accurate iden,fica,on of microorganisms is cri,cal to public health and safety and aZribu,on. Stakeholders need: •  Metrics and standards to

support rapid iden,fica,on and characteriza,on

•  Guidance that defines metrology for the field of microbiology

•  Methods valida#on for microbial iden#fica#on

www.in.gov hZp://www.mizozo.com

hZp://globaldefencemedia.com www.rosenkranz-‐lab.com

Sequence Based Microbial Iden,fica,on

Adapted from Welker and Moore 2011 Systema,c and Applied Microbiology

Whole Genome Sequencing

–  Whole genome sequencing is emerging as a rapid means to iden,fy an organism with strain, some,mes clone level resolu,on.

–  High resolu,on strain differen,a,on requires whole genome single nucleo,de polymorphism (SNP) analysis.

__

Reference

Single Nucleo,de Polymorphisms (SNPs)

Dele,on

Subs,tu,on

Inser,on

Microbial Genome

Importance of Accurate SNP Calling

•  Phylogeny of isolates from the 2011 Europe E. coli O104:H4 outbreak

•  Based on 20 SNPs –  Red and grey branches represent French and German isolates respec,vely without group strains indicated in blue.

Adapted from Grad YH et al. (2012) PNAS

SNP Calling Procedure

•  Strain Isola#on •  DNA extrac,on •  Library Prepara,on •  Sequencing •  Base calling •  Read processing •  Mapping •  SNP calling


•  Strain Isola,on •  DNA extrac#on •  Library Prepara,on •  Sequencing •  Base calling •  Read processing •  Mapping •  SNP calling


•  Strain Isola,on •  DNA extrac,on •  Library Prepara#on •  Sequencing •  Base calling •  Read processing •  Mapping •  SNP calling

Rothberg et al 2008 Nature


•  Strain Isola,on •  DNA extrac,on •  Library Prepara,on •  Sequencing •  Base calling •  Read processing •  Mapping •  SNP calling Rothberg et al 2011


•  Strain Isola,on •  DNA extrac,on •  Library Prepara,on •  Sequencing •  Base calling •  Read processing •  Mapping •  SNP calling

Base Calls Quality Score



Focus of this study Quality Trimming

Removing: low quality bases short reads trim long reads

Error correc,on correct predicted errors


•  Strain Isola,on •  DNA extrac,on •  Library Prepara,on •  Sequencing •  Base calling •  Read processing • Mapping •  SNP calling

Kalari et al 2012 Bioinforma,cs


Bayes Theorem GATK Variant Caller

McKenna et al 2010 Genome Research


Kalari et al 2012 Bioinforma,cs

Evalua,on of Read Processing

•  Perform whole genome sequencing – S. typhimurium LT2 – using Ion Torrent PGM

•  Read datasets –  Raw reads –  Trimmed based on quality score and length –  Error corrected

•  Compare read mapping and SNP calls –  True reference genome –  Two genomes with in silico muta,ons

Food Outbreak Relevant Strain

•  Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (S. typhimurium LT2) –  Gammaproteobacteria in the family Enterobacteriaceae –  Causes gasteroenteri,s and food poisoning

Year Source Infec#ons*

2012 Hedgehogs 14 / 6 /0

2011 Ground Beef 20 / 8 /0

2011 African Dwarf Frogs 241/72/0

2010 Teaching and Clinical Labs 109/13/1

2009 Water Frogs 85/29/0 hZp://www.lookfordiagnosis.com/

Outbreaks 2012-‐2009

*Cases / Hospital /Death Data from the www.cdc.gov

Salmonella typhimurium LT2 Genome

First sequenced in 2001 4.8 Mb Chromosome 94 kb plasmid

McClelland et al 2001 Science

Evalua,on of Read Processing •  Perform whole genome sequencing

–  S. typhimurium LT2 –  using Ion Torrent PGM

•  Read datasets – Raw reads – Trimmed based on quality score and length – Error corrected

•  Compare read mapping and SNP calls –  True reference genome –  Two genomes with in silico muta,ons

Quality Trimming Short Reads

Error Correc,on

Salamela and Schroder 2011 Bioinforma,cs

Evalua,on of Read Processing •  Perform whole genome sequencing –  S. typhimurium LT2 –  using Ion Torrent PGM

•  Read datasets –  Raw reads –  Trimmed based on quality score and length –  Error corrected

•  Compare read mapping and SNP calls – True reference genome – Two genomes with in silico muta#ons

In silico Mutated Genomes

•  Two ar,ficially mutated genomes – 480 variants and 5044 variants

•  Use evaluate the variant calling sensi,vity

ACGTGCACGTACGTCAC-GGACTTTACGACC ACGTGGACGTA-GTCACTGGACTTTACGACC

True Ref

Mutated Ref

Subs,tu,on Inser,on Dele,on

Mapping and SNP calling Read Dataset Read Processing Reference 480 known SNPs 5044 known SNPs

Raw Torrent Suite x x x

Raw TMAP + GATK x x x

Quality Trimmed TMAP + GATK x x x

Error Corrected TMAP + GATK x x x

TMAP – Torrent mapping algorithm hZps://github.com/iontorrent/TMAP GATK – Genome Analysis Toolkit (McKenna et al 2010 Genome Research)

Read Datasets •  Raw reads

–  2.2 M reads –  Read length mean 181 bp range 6 bp – 386 bp

•  Error correc,on using Coral –  CORrec,on using ALignment (Coral) –  Parameters: similar gap and mismatch penal,es –  2.2 M reads –  Read length mean 181 bp range 6 bp – 386 bp

•  Read Trimming –  Parameters

•  Size cutoffs: 100 – 350 bp •  Quality trimming: mean 99% accuracy as defined by the instrument

–  1.7 M reads –  Read length mean 205 bp range 100 bp – 349 bp

Chromosome Plasmid

65

70

75

80

110

130

150

170

0 1e3 2e3 3e3 4e3 5e3 0 2.5 5.0 7.5Genome Position (Kb)

Cov

erag

e Data SetError Corrected

Quality Trim

Raw

Comparable coverage paZern for all data sets

Mapping Coverage

Similar coverage paZern for mutated and true reference genomes and mapping algorithms

Comparison of SNP Call Abundance Reference 480 SNPs 5044 SNPs

0

2000

4000

6000

Raw Qual Error Raw Qual Error Raw Qual ErrorRead Dataset

SNP

Cou

nts

Mapping Algorithm and SNP Caller

Torrent Suite

TMAP + GATK

Distribu,on of SNP Call Quality Scores

Raw Quality Trim Error Corrected

0200400600

0200400600

0200400600

Reference

480 SNPs

5044 SNPs

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4SNP Quality Score (1000×)

SNP

coun

t

Error Corrected SNPs have lower quality scores than introduced SNPs

SNP call sensi,vity for introduced variants

480 known SNPs Raw Quality Trim Error Correc#on Torrent Suite

True Posi,ve 411 410 454 464 False Nega,ve 69 70 26 16

5044 known SNPs Raw Quality Trim Error Correc#on Torrent Suite

True Posi,ve 4403* 4403* 4814 4809

False Nega,ve 641 641 230 234

* Not the same variants

Both Error Corrected and the Torrent Suite have the highest sensi,vity

Conclusion

•  Use of error correc,on method increases coverage but may adversely affects SNP calling specificity

•  Future work to develop reference datasets and analysis tools for use in algorithm tes,ng and pipeline valida,on

Acknowledgements

•  Team: Dr. Jayne Morrow (leader), Sandra Da Silva, and Lindsay Vang

•  Jenny McDaniel, Jus,n Zook and Steve Lund

Contact Informa,on: Nathan Olson [email protected]

Disclaimer"

Material Measurement Laboratory

Certain commercial equipment, instruments, or materials are iden9fied in this presenta9on in order to specify the experimental procedure adequately. Such iden9fica9on is not intended to imply recommenda9on or endorsement by the Na9onal Ins9tute of Standards and Technology, or affiliated venues nor is it intended to imply that the materials or equipment iden9fied are necessarily the best available for the purpose. All opinions expressed in this presenta9on are the authors’ and do not necessarily reflect the policies and views of NIST or affiliated venues.

Evaluation of the impact of error correction algorithms on SNP calling.

Science

Transcript of Evaluation of the impact of error correction algorithms on SNP calling.