Computational protocol: Application of whole genome re-sequencing data in the development of diagnostic DNA markers tightly linked to a disease-resistance locus for marker-assisted selection in lupin (Lupinus angustifolius)

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Protocol publication

[…] The nine re-sequenced cultivars were Unicrop (the first fully domesticated cultivar in this species which was release in 1973), Yorrel (released in 1989), Merrit (1991), Kalya (1996), Tallerack (1997), Quilinock (1999), Mandelup (1994), Coromup (2006), and Jenabillup (2007). Re-sequencing of the nine cultivars was performed by the whole genome shotgun (WGS) approach []. DNA was extracted from three-week-old seedlings grown in a glasshouse. DNA was randomly sheared by nebulization, end-repaired with T4 DNA polymerase, and size-selected by gel electrophoresis on 1 % low-melting-point agarose. A sequencing library of insert-size 500 bp was constructed for each cultivar according to the Illumina Inc. manufacturer instructions. Pair-end sequencing of the sequencing libraries was performed on NGS platform Hiseq2000 at Beijing Genome Institutes (BGI-Shenzhen). The sequencing data for each cultivar were assembled by SOAP de novo []. The assembled sequences were aligned into corresponding scaffolds based on the reference draft genome sequence of Tanjil by Short Oligonucleotide Alignment Program (SOAP 2.20) []. [...] Genome sequence data of the nine re-sequencing cultivars were mapped onto the reference sequences originated from cultivar Tanjil []. Based on the mapping result by SOAP 2.20, uniquely mapped single-end and paired-end results were used in the SNP calling. The genotypes of each individual at every genomic site were calculated by SOAPsnp []. Polymorphic loci against the reference sequence were selected and then filtered. SNP markers were recorded if they are supported by at least 3 reads with quality value greater than 20. The InDel markers (insertions and deletions shorter than 10 bp) were identified by gap allowed alignment (additional parameter of “-g 10” was used in SOAP2). InDels supported by at least three pair reads were detected by SOAPindel pipeline (http://soap.genomics.org.cn/) as described by Zheng et al []. Genomewide genetic diversity between reference cultivar Tanjil and the nine re-sequenced cultivars was based on the calculation of SNP abundance along each linkage group in the genetic map []. SNP numbers were counted in each non-overlapping 100 kb interval and displayed in a circular histogram using the software of circus (http://circos.ca/). […]

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