The SMRT sequencing from Pacific Biosciences (PacBio) is a third-generation ‘sequencing-by-synthesis’ approach which overcomes shortcomings of first- and second-generation sequencing approaches. The SMRT sequencing is cost-effective, time-efficient, and computationally simple approach. However, the unique strength of SMRT sequencing method relative to other next-gen sequencing methods is single-molecule reads that exceed 10 kb in length. Although this system has an inherent sequencing error rate, the errors are random and can be overcome by redundancy.
Herein, a protocol of PacBio SMRT library construction was developed. Six and eight barcodes were added to each forward and reverse primer respectively, thus genotyping 48 accessions simultaneously with a primer pair. These barcodes were found efficient to identify all 48 accessions in a library. The DNA and primers were distributed in 96 well plate such that two libraries were prepared together for one gene. After PCR concentration of each amplicon was determined using capillary electrophoresis and agarose gel electrophoresis. A standard of measuring concentration of each amplicon and mixing all 48 amplicons in a library according to relative concentrations was optimized. The concentrations of all COBRA Like genes within a library were also measured and mixed accordingly into a one Eppendorf tube. Three replicates of each library were sent to company for PacBio-SMART sequencing. Total 25 genes were included in the library. The Python script was developed to segregate sequences and identify haplotypes from the pool of SMRT sequences. This protocol can be used for haplotyping complex gene families in other polyploid crops with large and complex genome.