Simple procedure for haplotype sequencing from diploid samples

The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Cells were serially diluted to a concentration of one cell per microlitre by counting with a haemocytometer at each dilution. One microlitre samples, each potentially containing a single cell, were lysed with KOH and flash frozen. The lysates were neutralised and divided into six aliquots (except for the sperm samples which were not divided into aliquots). A test phi29 polymerase amplification was undertaken on one aliquot from each sample. Less than 50% of test amplifications were positive indicating that the dilution of cells was greater than predicted and that therefore it was unlikely that any sample contained more than one cell. The remaining five aliquots from each positive sample were phi29 amplified and libraries were prepared and sequenced for each. Contigs were obtained by mapping to the bovine UMD3.1 reference genome assembly and scaffolds were assembled by joining adjacent contigs that were within a predetermined threshold distance of each other. Scaffolds that appeared to contain artefacts of CNV or repeats were filtered out leaving scaffolds with an N50 length of 48kb and a 98.9% genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 141kb but only ~56% genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing.