CARS at ICOPA XII, August 2010

Next-gen. Haemonchus contortus genomics

The life cycle of H. contortus

Refs.: Gasser, R.H., unpub. review; Nikolaou and Gasser (2006), Int. J. Parasitol. 36, 859-868;Prichard and Geary (2008), Nature 452, 157-158.

C. elegans

C. briggsae

C. japonica

C. remanei

C. brenneriE

lega

ns

Ca

en

orh

ab

ditisC. sp. 3 PS1010

C. drosophilae

C. sp. 1 SB341

Dro

so

-p

hila

e

Brugia malayi

Pristionchus pacificus

Meloidogyne hapla

Meloidogyne incognita

H. contortus' evolutionary context

Heterorhabditis bacteriophora

Haemonchus contortus

Protein and DNA functions are conserved

Refs.: Rufener et al. (2009), PLoS Pathog. 5, e1000380 ; Hu et al. (2010), Biotechnol. Adv. 28, 49-60.

*MonepantelR mutation

*

*

Ce-ant-1.1

Hc-ant-1.1

*

Next-generation sequencing in a nutshell

Ref.: Miller et al. (2010), Genomics 95, 315-327.

Assemble DNA reads

Map RNA reads onto assembly

Scaffold contigs with RNA

ERANGE (Bowtie + BLAT)

RNAPATH

Final assembly

Repeat once

Filter out E. coli contigs

Velvet

Assembly strategy used for C. sp 3 PS1010

RNA-mediated scaffolding of Velvet genomic supercontigs

(RNAPATH)

Within-supercontig RNA-seq reads

RNA-seq exons

Velvet genomic supercontigs

Velvet+RNAPATH supercontigs

Cross-supercontig RNA-seq reads

RNA scaffolding

PS1010 assembly statistics

Assembly Total (Mb)

Super-contigs

Max. sc.size (kb)

N50 (kb)

Pred. genes

Genomic Velvet(k = 47)

79.8 44.9K 45.7 5.1 27,741

Genomic Velvet + RNAPATH

x2

79.8 33.6K 96.3 9.4 22,851

cDNA Velvet 17.8 27.9K 14.5 1.05 n/a

Genome size: 100 Mb [?]. Est. genomic coverage: ~170x.An additional 4.6 Mb of Velvet supercontigs matched E. coli.

H. contortus (McMaster) assembly statistics

Assembly Total (Mb)

Super-contigs

Max. sc.size (kb)

N50 (kb) Pred. genes/pe

ptides

Genomic Velvet(k = 37)

272.3 440K 27.9 1.26 n/a

Genomic Velvet + RNAPATH[L1 cDNA]

272.3 425K 45.7 1.37 n/a

Genomic Velvet + RNAPATH[L2 cDNA]

272.3 421K 43.1 1.40 n/a

cDNA Oases 67.6 132K 22.5 0.92 86,671

Genome size: ~290 to 340 Mb. Est. genomic coverage: ~35x.

Only 40% of reads map to the assembly

Reads mapped with bowtie by Titus Brown.

Of reads which did map, 50% went to ≥1.8 kb contigs.

What is to be done?

1. Brute force: push coverage up to at least 50x.

Latest round of sequencing just raised it to ~47x.

2. Larger insert sizes: current libraries are ≤325 nt.

~500 nt goal; also, try jumping libraries (after PS1010 test).

3. Work smarter: remove erroneous or highly repetitive reads.

New method for removing low-freq. 32-mers from Brown et al.:

http://ivory.idyll.org/blog/jul-10/kmer-filtering.html

http://ivory.idyll.org/blog/jul-10/illumina-read-phenomenology.html

Thanks:

Robin Gasser Isolated genomic DNABronwyn Campbell Isolated stage- and sex-specific RNANeil Young Aided DNA and RNA work

Ali Mortazavi Devised RNAPATH; earlier H. contortus assembly

Brian Williams cDNA library constructionLorian Schaeffer Illumina sequencingIgor Antoshechkin Optimized sequencing protocols

Titus Brown k-mer filteringJason Pell, Adina Chuang

Jacobs Genome Center Infrastructure and fundingARC, NIH, and HHMI