Targeted genomic enrichment and

SMRT sequencing of immune-related

gene complexes

John Hammond

(john.Hammond@pirbright.ac.uk)

Innate immune gene variation: germ-line

encoded NK cell receptors and MHC class I

Haplotype variation, high polymorphism and variegated expression creates NK

cell subsets with different specificities and functionsLRC NKC

MHC

Genetically defined and inbred animals are key to dissecting genomic

function

This arm of the immune system is critical in controlling and resolving viral

infection

Diverse NK cell receptor systems are rapidly evolving under intense

selection pressure from rapidly evolving pathogens.

CD8 T cells

• Reference genome not well resolved in these regions

• Poor SNP coverage in hard to assemble repetitive

regions

• Reference genome presents only one haplotype of

many

Highly repetitive regions are difficult to

sequence with short read technology

Human MHC class I is highly diverse but

haplotypes do not vary in gene content

Gene A B C E F G

Alleles 4,200 5,091 3,854 27 30 60

Proteins 2,923 3,664 2,644 8 5 19

Nulls 200 150 144 1 0 3

A greater degree of

structural variation

in cattle

Sequence identity comparison of two

cattle class I genomic haplotypes

MHC class II

The current HD SNP chip does not interrogate

MHC variation

The cattle KIR complex has expanded and demonstrates all

the features of a functional immune complex

Key properties of KIR loci Human KIR Cattle KIR

Inhibitory and Activating

Activating genes disarmed

Functionally variable haplotypes ?

Polymorphic

Paired activating and inhibitory

receptors

Identity

The cattle NKC is largely correct in reference assembly-

determined by BAC clones and a new cattle reference assembly

Schwartz et al. 2017. Immunogenetics.

Dis

tan

ce

be

twe

en

SN

Ps

SNP position in the genome

~280 kb

8 SNPs & 17 genes

The cattle natural killer complex

• missing SNP variation over the most diverse region

The identity between genes and gene blocks is too

high to map short reads over the KLRC region

% o

f re

ads d

iffe

rent

from

UM

D3.1

Location on BTA5

Illumina 250 bp PE reads

Enrichment and de novo assembly of

immune related gene complexes in cattle for

SNP discovery.

Cattle are arguably the most important livestock species: they

provide humans with meat, milk, hides, traction, manure, status

and security.

Reducing the burden of disease can have enormous positive

impact for food security and welfare.

Complex immune traits are phenotypically diverse making

breeding/selection processes challenging-but there are many

opportunities!

Where are the immune genes known to be involved bTB?

Prof Liz Glass, Roslin.

Used the Roche (Nimblegen) SeqCap EZ system

Targeted enrichment of Immune-related gene

cluster with Roche Nimblegen probes

Library prep needed considerable optimisation

Average pull down fragment was 5.5 kb

http://www.google.co.uk/url?sa=i&rct=j&q=Nimblegen+tiling&source=images&cd=&cad=rja&docid=RqmqtKzR_JO9XM&tbnid=NG3OIJhNAfRi9M:&ved=0CAUQjRw&url=http://www.nimblegen.com/choice/&ei=KSHoUbi5FsOVO5v7gbgG&bvm=bv.49478099,d.ZWU&psig=AFQjCNG3w84UM3ozPlZoopmCwGwvKHeulQ&ust=1374253620612261http://www.google.co.uk/url?sa=i&rct=j&q=Nimblegen+tiling&source=images&cd=&cad=rja&docid=RqmqtKzR_JO9XM&tbnid=NG3OIJhNAfRi9M:&ved=0CAUQjRw&url=http://www.nimblegen.com/choice/&ei=KSHoUbi5FsOVO5v7gbgG&bvm=bv.49478099,d.ZWU&psig=AFQjCNG3w84UM3ozPlZoopmCwGwvKHeulQ&ust=1374253620612261

Four rounds of probe design and optimisation

Illumina set

First design to use “masking” as a way to deal with multiple variant targets for single

genomic region, thus reducing over-capture of non-variant subregions.

NG1-Pilot set ~ 9Mb with 50 known matches

Update included an increase number of target regions and increase in number of variant

inputs per region. Match level stretched to 50 for coverage.

NG2 ~ 5Mb with 50 known matches

Similar strategy to 151221_BTAU_TPI_NiGen2_EZ_HX3. Size of NKC target decreased,

and MHCIIb,TPI, RP and IG regions removed.

NG3 ~ 5Mb Main aim is to reduce off target mapping of 70-80 %

Redesign of 170131_BTAU_DH_TPI_EZ_HX3. Match levels reduced to 3, mapping targets

against reference included, efficiencies estimated, probes in NKC region replicated 3x and

MHC replicated 2x.

2 animals

PacBio sequencing; 23 animals

NG1 probe set- good enrichment but still much off target

Custom chromosome

NKC

MHC

Custom chromosome

NKC

MHC

NG2 probe set- better enrichment but NKC dropped out

Probe performance NKC (NG2)

-

20,000,000

40,000,000

60,000,000

80,000,000

100,000,000

120,000,000

chr5/NKC

Blue = nucleotides binding to whole chromosome

Green = nucleotides binding to target area

Probe performance LRC (NG2)

-

20,000,000

40,000,000

60,000,000

80,000,000

100,000,000

120,000,000

chr18/LRC

Blue = nucleotides binding to whole chromosome

Green = nucleotides binding to target area

-

50,000,000

100,000,000

150,000,000

200,000,000

250,000,000

chr23/MHC

Probe performance MHC (NG2)

Blue = nucleotides binding to whole chromosome

Green = nucleotides binding to target area

• On-target efficiency sacrificed for overlapping probe

coverage- not entirely necessary

• Many off-target regions unsupported by probe

sequence- multimapping and polymorphism/variation

• Masking does not adequately reduce the redundancy

of the inputs but does allow probes with similar

sequences to hybridize to similar haplotype

sequences resulting in greater depth of coverage

Probe design summary

• At least subreads from 2 SMRTcells combined for de

novo assembly with Canu

• filtered subreads as input

• Default parameters minreadlength>1kb• for MHC minreadlength >3kb improves assembly

• For the LRC this does not improve the assemblies

• gfa file as output from Canu screened for contigs that

contain MHC or KIR genes/haplotypes with bandage,

which were then extracted and mapped to known

MHC/KIR haplotypes

Enrichment and De novo assembly

A18 – “gene 6” reconstructed haplotype

252NC1 TRIM26

Gene6

6*01301

2 contigs: 170kb, 53kb

105991

9 contigs

NC1 TRIM26

Gene6

6*01301

705983TNC1 TRIM26

Gene6

6*01301

7 contigs

A31 – “gene 1+2” reconstructed haplotype

NC1 TRIM26Gene1

*02101Gene2

*02201

20005

8 contigs

604652NC1 TRIM26

Gene1

*02101Gene2

*02201

8 contigs

Heterozygous A18/A31(mixed reads from 252 and 200005 as input in de novo)

• Shared regions assemble contigs that are more similar to the haplotype with

more reads

• Alleles for gene 6, gene 2, gene 1 identical to previous

• FALCON

MHC class I full-length bovine haplotypes

P3 NC1 1 4 2 6 T

TRIM26

A14

ARS14 P3 NC1 5 2 T

35kb 62kb 67kb 69kb 58kb103kb

A11 3 2 T

Angus P3 NC1 3 2 6 TP6

Brahman P3 3 2 T3

A18

A31

P3 NC1 6 T

P3 NC1 1 2 T

20kb

Breed IDknown

haplotypede novo

haplotypeallele

haplotype allelesHereford Dominette ? 02*07001; 05*07201

Friesian 252 A18 A18 A18 06*01301

Friesian 200005 A31 A31 A31 01*02101; 02*02201

Friesian 105991 A18 A18 A18 06*01301

Friesian Herman A14/? A14? 01*02301; 04*02401;02*02501; no 06*

Friesian 505204 A14 A14 A14? 01*02301;04*02401;02*02501; no 06*

Friesian 604652 A31 A31 A31 01*02101; 02*02201

Hereford Domino ? 02*06001;06*04001;05*07201

Friesian 705983T A18 A18 A18 06*01301

Highland 8052 ? new? 01*03102; new02*?

Sahiwal 83H ? new? new03*?

Friesian 206818 ? A14/A14 A14 01*02301;04*02401;02*02501;06*04001

Friesian 706886 ? A14? new01*;02*02501;04*02401

Friesian 206846 ? new? 01*01901;02*02501

Friesian 206853 ? A14/het? A14/? 01*02301;04*02401;02*02501;06*04001

Friesian 504805 A10/A14 01*02301;04*02401;03*00201; new02*

Friesian 504805 A10/A1401*02301; 04*02401;02*02501;

03*00201;

Friesian dried706823 ? new? 01*02101;04*02401;02*02501;

Friesian 306812 ? new? new 01*;02*00801;04*02401

Friesian 159 A31 A31 01*02101; 02*02201

Most likely haplotype based on alleles

1-3bp differences to allele

• 200005 – reads from 2 SMRTcells* (>940,000 subreads, >1kb length)

200005_NG1+Sequel

KIR haplotype contains block A and B

• 200005 – reads from 2 SMRTcells* (>625,000 subreads, >3kb length)

*includes one Sequel run

• 252 – reads from 4 SMRTcells* (>863,000 subreads with >3kb length):

8 contigs, longest 118kb

KIR haplotype from 252 missing block B?

252_NG1

• 252 – reads from 1 SMRTcells (>486,000 subreads >1kb length):

8 contigs

One contig*includes one Sequel run

De novo assembly of KIR from two other A18 animals

705983T_NG2+2rep

105991_NG2+2rep

• 705983T – reads from 2 SMRTcells (> 776,000 subreads >1kb length)

• 105991 – reads from 2 SMRTcells (> 606,000 subreads >1kb length)

• Also missing block B?

- Illumina reads from 125 Holstein bulls mapped to

immune-related genecluster haplotypes (BWA)

- NKC, LRC, MHC

- SNPs called with x variant caller

- Filtered SNPs: QUAL > 900, strictly biallelic (no INDELs)o Called for all individuals; Alternative allele frequencies: between 5%

and 95% (only NKC)

- Selected SNPs 10-15kb apart across region and based

on representation of mapping data

- checked 50bp flanking region if repeat within haplotype

o Transferability to other haplotypes (according to SNP

coordinate) checked

o SNPs checked for haplotype specificity, and gene specificity

(MHC only)

SNP selection using de novo assembled

haplotypes

New SNP panel over 3 different gene complexes being

used to increase the power of GWAS for complex

disease traits

The single SNP on the current Illumina SNP chip

The cattle new cattle LRC SNP panel

UMD3.1

A14

A11

A18

A31

pink SNPs

blue SNPs

orange SNPs

MHC haplotype SNP selection

~280 kb

MAGOHB

KLRA KLRJKLRC1-3

NKC haplotype SNP selection

First round of SNP selection successful

(~70 % success).

Established segregating

markers in a cohort or 1500

extreme phenotype bTB

resistant cattle, currently doing

GWAS

Acknowledgments

Immunogenetics

Nick Sanderson

Alasdair Allan

Mark Gibson

John Schwartz

Rebecca Philp

Clare Grant

Karen Billington

Paul Norman

Libby Guethlein

Peter Parham

Farbod Babrzadeh

John Young

Richard Borne

Doro Harrison

Elizabeth Morecroft

Kevan Hanson

William Mwangi

Giuseppe Maccari

Derek Bickhart

Timothy Smith

William Thompson

Juan Medrano

Denise Raterman

Cynthia Moehlenkamp

George Mayhew

BB/M027155/1, BB/J006211/1

GCRF Databases and ResourcesLiz Glass