Coverage-based consensus calling (CbCC) of short sequence reads and comparison of CbCC results to identify SNPs in chickpea (Cicer arietinum; Fabaceae), a crop species without a reference genome.
OBJECTIVE
Next-generation sequencing (NGS) technologies are frequently used for resequencing and mining of single nucleotide polymorphisms (SNPs) by comparison to a reference genome. In crop species such as chickpea (Cicer arietinum) that lack a reference genome sequence, NGS-based SNP discovery is a challenge. Therefore, unlike probability-based statistical approaches for consensus calling and by comparison with a reference sequence, a coverage-based consensus calling (CbCC) approach was applied and two genotypes were compared for SNP identification.


METHODS
A CbCC approach is used in this study with four commonly used short read alignment tools (Maq, Bowtie, Novoalign, and SOAP2) and 15.7 and 22.1 million Illumina reads for chickpea genotypes ICC4958 and ICC1882, together with the chickpea trancriptome assembly (CaTA).


RESULTS
A nonredundant set of 4543 SNPs was identified between two chickpea genotypes. Experimental validation of 224 randomly selected SNPs showed superiority of Maq among individual tools, as 50.0% of SNPs predicted by Maq were true SNPs. For combinations of two tools, greatest accuracy (55.7%) was reported for Maq and Bowtie, with a combination of Bowtie, Maq, and Novoalign identifying 61.5% true SNPs. SNP prediction accuracy generally increased with increasing reads depth.


CONCLUSIONS
This study provides a benchmark comparison of tools as well as read depths for four commonly used tools for NGS SNP discovery in a crop species without a reference genome sequence. In addition, a large number of SNPs have been identified in chickpea that would be useful for molecular breeding.
Centre of Excellence in Genomics, International Crops Research Institute for the Semi-Arid Tropics, Patancheru 502324, Andhra Pradesh, India.
Sarwar
S
Azam
Vivek
V
Thakur
Pradeep
P
Ruperao
Trushar
T
Shah
Jayashree
J
Balaji
BhanuPrakash
B
Amindala
Andrew D
AD
Farmer
David J
DJ
Studholme
Gregory D
GD
May
David
D
Edwards
Jonathan D G
JD
Jones
Rajeev K
RK
Varshney
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods; D029863:Cicer / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D016384:Consensus Sequence; D018556:Crops, Agricultural / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D014644:Genetic Variation; D018745:Genome, Plant; D005838:Genotype; D020641:Polymorphism, Single Nucleotide; D012015:Reference Standards; D015203:Reproducibility of Results; D016415:Sequence Alignment / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D059467:Transcriptome
D018744:DNA, Plant