nextera tools for microbial...

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Developing Nextera tools for microbial

sequencing

Niall Gormley 17th May 2013

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No. 1 guiding principle of sample prep is…

Simplicity

No. steps Tube transfers Volumes

Avoid gels Avoid PCR

Compromise by App

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A-tail

Clean up

Ligate adaptors

PCR

Shear

Clean up

2x Clean up

Gel

Clean up

End polish

~ 1 day

1ug

TruSeq DNA

A-tail

Ligate adaptors

Shear

End polish

Bead size select

2x Clean up

Clean up

½ day

1ug

TruSeq DNA PCR-Free

Tagment

Clean up

Clean up

PCR

1.5 hr

50ng

Nextera

Tagment

Clean up

PCR

1.5 hr

1ng

Nextera XT

4

Tagment

Clean up

Nextera PCR free?

10 min?

? ng

Tagment

Clean up

PCR

1.5 hr

1ng

Nextera XT

3’

+ 5’ 3’

3’ 5’

Rd1 SP

Rd2 SP’

Challenges:

More steps

Transposome

Indexing

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The next-gen ‘Agar plate’:

vs

Bta colonies (102-103 per plate)

• Very cheap • Simple

Clusters (1Million / mm2) • Turn-key solution / multiple pathogens • ‘see it all’ • >>Greater detail • Info at the ultimate resolution

Nextera prep

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Some examples of ultimate resolution

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Both: smear +ve for Tuberculosis 14 days apart

Culture +ve sputum samples tested at nat ref lab

2 students overlap briefly at Cambridge language school

Resistance to: • Isoniazid

• Prothionamide

• Streptomycin

Strain ID: • Beijing

• MIRU-VNTR

50ng DNA - Nextera – MiSeq – 150bp reads – Two isolates genetically identical – No SNP differences in mapped genes

Analyzed 35 genes/promoters associated with resistance to antibiotics – C-15T mutation in inhA (shared target for Iso & Pro) – V77G mutation in gidB (putative 16s rRNA M’tase – confers low-level resistance to Strep)

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Outbreak commences in neonatal ward

Same ward before outbreak Rest of hospital

Initial antibiogram suggest diff to ST22 (EMRSA-15)

Outbreak ward

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Sequenced 14 isolates

Same ward before outbreak Rest of hospital

Outbreak ward

All strains on outbreak ward are ST 22

4 strains have different ST type

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Sequencing reveals 2 distinct groups

reference

Maximum likelihood tree

Separated by 102 SNPs

Hypermutator

Outbreak ward

Identified Hypermutator with frameshift in mutS

Single A insertion in hemG causing truncation of HemG

100% concordance between resistance phenotype and mutations identified in the sequence genomes Small colonies

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Single read 36 base yielding 27 million reads

81%: 170x coverage of E.coli genome

10%: 37,000x coverage of pUC19

9%: F plasmid and pUC19 insert

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Extreme sample prep =

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Biotin

Biotin

Biotin

Biotin

Mate-pairs

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Shear

End polish

Cap linker

Circularize

EcoP15I digest

Biotin enrich

End polish/A-tail Clean up

Ligate adaptors

PCR

Exonuclease

Clean up

EtOH ppt

Clean up

Clean up

Clean up

Bead washes

Bead washes

Gel

Gel Clean up

Methylation

Clean up Gel

Clean up

2006

Fill in

Shear

End polish

Circularize

Shear

End polish

Ligate adaptors

emPCR

Exonuclease

Clean up

Bead size cut

Clean up

Clean up

Clean up

Clean up Hairpin linker

Clean up

Clean up

Methylation Clean up

Cleave hairpin

Clean up

2009 Shear

End polish

Cre-Lox linker

Fill in

Circularize

EcoP15I digest

Biotin enrich End polish

A-tail

Ligate adaptors

PCR

Exonuclease

Clean up

Bead size cut

Bead size cut

Clean up

Clean up

Clean up

Bead washes

Bead washes

Bead washes

Gel Clean up

2011

Clean up

Manipulation of very large DNA molecules

Basic short insert preps

• Gels • Clean-ups • Ligations

Challenges

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Bio Bio

+

XmnI

Bio Bio

XmnI

Bio Bio

Strep Strep Bio Bio

Ligate

Digest

Strep gel shift

Bio

Strep - + - + - +

Bio

65% ligation efficiency at short end => 42% efficiency both ends

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Shear

End polish

Cap linker

Circularize

EcoP15I digest

Biotin enrich

End polish/A-tail Clean up

Ligate adaptors

PCR

Exonuclease

Clean up

EtOH ppt

Clean up

Clean up

Clean up

Bead washes

Bead washes

Gel

Gel Clean up

Methylation

Clean up Gel

Clean up

2006

Fill in

Shear

End polish

Circularize

Shear

End polish

Ligate adaptors

emPCR

Exonuclease

Clean up

Bead size cut

Clean up

Clean up

Clean up

Clean up Hairpin linker

Clean up

Clean up

Methylation Clean up

Cleave hairpin

Clean up

2009 Shear

End polish

Cre-Lox linker

Fill in

Circularize

EcoP15I digest


A-tail

Ligate adaptors

PCR

Exonuclease

Clean up

Bead size cut

Bead size cut

Clean up

Clean up

Clean up

Bead washes

Bead washes

Bead washes

Gel Clean up

2011

Gel

Shear

Biotin end polish

Circularize

Clean up

Clean up

Exonuclease

Clean up

Bead washes


A-tail

Ligate adaptors

PCR

Shear

Bead washes

Bead washes

Gel

2008

Clean up

Clean up

Biotin

Bio-dNTPs

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Strand displace Bead size cut

Clean up

Circularize

Tagment

Bead washes


A-tail

Ligate adaptors

PCR

Exonuclease Shear

Bead washes

Bead washes

Clean up

2013

Gel

Shear

Biotin end polish

Circularize

Clean up

Clean up

Exonuclease

Clean up

Bead washes


A-tail

Ligate adaptors

PCR

Shear

Bead washes

Bead washes

Gel

2008

Clean up

Biotin

Bio-dNTPs

Biotin

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Bacterial de novo assembly with Gel-Free Mate-Pair

E.coli de novo Assembly Metrics

Total size [bp] 4,664,930 (15)*

N50 scaffold [bp] 4,635,607 (1)*

Max scaffold [bp] 4,635,607

E.coli mate-pair data - 2 x 100 bp ( 1 of 4 Indexed bacterial libraries on a single MiSeq Run)

* Number of contigs in data set

Complete de novo assembly

Median size 3971 bp Total Reads 1,262,185 % mate-pairs 98.2 % % Unique 99.8 % Diversity 338,674,000

Mate-Pair Protocol + MiSeq = DNA to de novo in 3 days!

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Size GC content N50 Max

Scaffold Total size Genome Coverage

Input Coverage

E. coli DH10B 4.7 M 50% TruSeq 109,035 (13) 330,951 4,544,106 (136) 95% 59

Mate-Pair 3,690,672 (1) 3,690,672 4,762,657 (68) 96% 57 R. sphaeroides 4.6 M 67%

TruSeq 279,376 (6) 551,032 4,161,361 (73) 82% 47 Mate-Pair 2,590,288 (1) 2,590,288 4,591,572 (64) 94% 31

B. cereus 5.4 M 35%

TruSeq 485,654 (3) 1,197,538 5,371,324 (70) 92% 66 Mate-Pair 3,611,328 (1) 3,611,328 5,444,030 (39) 99% 40

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Mate pairs

WGS paired ends

Size selected

Mate pair Input

Contigs

Scaffolds

N50 contig

N50 scaffold

Ilmn + JGI Gel free 1ug 27 4 408.4 kb 3,493 kb Ilmn + JGI ~6 kb (gel) 4ug 29 6 275.1 kb 3,692 kb JGI + JGI ~ 10 kb (gel) >10ug 48 8 188.2 kb 4,060 kb

Ilmn + JGI Gel free 1ug 8 1 744.3 kb 6,328 kb JGI + JGI ~ 10 kb (gel) >10ug 7 1 1,190.2 kb 6,355 kb

Cellulomonas flavigena

74% GC

Conexibeater woesei 73% GC

2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 Fragment size kb

Fragment size kb

Num

ber o

f rea

ds

Num

ber o

f rea

ds

C. flavigena C. woesei

JGI JGI Ilmn 1ug

Ilmn 1ug

Ilmn 4ug

@JGI

Data courtesy of Feng Chen

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Clean up

Circularize

Tagment

Bead washes


A-tail

Ligate adaptors

PCR

Exonuclease Shear

Bead washes

Bead washes

Clean up

2013

Biotin Biotin


Clean up

Circularize

Tagment

Biotin enrich PCR

Exonuclease Tagment

Clean up

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Shear Tagment

E.coli Human

Ec Hu

Shear Tagment

Human E.coli

Ec Hu

Combine &

Split

Ec Hu Ec Hu

Combine &

Split

Ec Hu Ec Hu

Shear Tagment

Ec Hu Ec Hu

Shear Tagment

Hu Ec Hu Ec

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Shear Shear

Tagment Shear

Shear Tagment

E.c

oli

Hum

an

% Paired ends 3.4 29.1 1.7 61.5 % Mate Pairs 95.1 69.6 95.8 35.3 Diversity 159 M 49 M 5,346 M 127 M

% Paired ends 4.4 34.4 0.7 47 % Mate Pairs 90 60. 94 45 Diversity 4 M 50 M 2,947 M 218 M

Tagment Tagment

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0

2000

4000

6000

8000

10000

12000

14000

0 2500 5000 7500 10000 12500 15000

1000 ng

500 ng

250 ng

125 ng 62.5 ng

E. coli MG1655 N50 Max Scaffold Total size Genome Coverage

Input Coverage

1000 ng 4,639,989 (1) 4,639,989 4,672,244 (78) 99.4% 180 500 ng 4,617,536 (1) 4,617,536 4,652,083 (84) 99.1% 163 250 ng 4,631,394 (1) 4,631,394 4,661,881 (67) 99.5% 140 125 ng 4,644,837 (1) 4,644,837 4,689,356 (86) 99.6% 158 62.5 ng 4,648,614 (1) 4,648,614 4,684,742 (65) 99.5% 153

Lastly: in the hands of R&D

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Thanks!

nextera tools for microbial...

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