Targeted Sequencing and De Novo Assembly of a Highly Repetitive Gene Family with Long PacBio Reads of Inserts - Potato

We have established RenSeq as a technique to capture, sequence and study nucleotide binding and leucine-rich repeat (NLR) encoding plant disease resistance genes (Jupe et al. 2013).  Following targeted enrichment and sequencing with Illumina MiSeq we could annotate and de novo assemble whole NLR complements of several Solanaceae (Jupe et al. 2013, Andolfo et al. 2014). Precise de novo assembly of all NLRs with MiSeq reads is however hampered by multiple paralogues and the size of this gene family. To overcome this problem, we updated our RenSeq pipeline to utilize long fragment sequencing on the PacBio RSII platform. We could generate 2-5kb high quality reads, some of them containing full length NLR genes. De novo assembly of this dataset allowed the individual assembly of close paralogues, previously not possible with MiSeq data. Moreover, we could assemble up to 2kb of promoter and terminator regions, and a 35 kb cluster harboring five Sw5 paralogs. Combined with genetic data from populations segregating for resistance against Phytophthora infestans and P. capsici, we identified candidate resistance genes from the de novo assembled NLRs. Amplification and cloning of full length co-segregating NLRs was carried out and these are currently functionally tested.

RenSeq in combination with PacBio allows the accurate de novo assembly of highly similar NLR gene family members and the capture of up to 2kb flanking regions.

Jupe et al. 2013 The Plant Journal, 76: 530-544

Andolfo et al. 2014 BMC Plant Biology, 14: 120