Abstract
BackgroundAlmost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and consequently what is published is a composite of the two. This means that the relationship between alternate alleles at two or more linked loci is lost. We have developed a simplified method of directly obtaining the haploid sequences of each genome copy from an individual organism.ResultsThe diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Samples were: 1) lymphocytes from a single Angus steer; 2) sperm cells from an Angus bull; 3) lymphocytes from East African Zebu (EAZ) cattle collected and processed in a field laboratory in Eastern Kenya. Haploid sequence from a fosmid library prepared from lymphocytes of an EAZ cow was used for comparison. 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 and divided into six aliquots (except for the sperm samples which were not divided into aliquots). Each aliquot was amplified with phi29 polymerase and sequenced. 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 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 27–133 kb and a 88–98 % genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97–201 kb but only ~27–68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data.ConclusionsWe have developed a set of laboratory protocols and software tools that will enable any laboratory to obtain haplotype sequences at only modestly greater cost than traditional mixed diploid sequences.
Highlights
Almost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and what is published is a composite of the two
It is becoming increasingly apparent that a substantial amount of the phenotypic variation within populations is attributable to rare genomic variants [2] and the haplotypes on which these variants lie cannot be reliably inferred from genotypes at common marker single nucleotide polymorphisms (SNP)
We have developed a single cell isolation protocol that allows the accurate selection of appropriately diluted DNA whilst minimizing the number of pipetting steps that might shear the DNA
Summary
The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Each potentially containing a single cell, were lysed and divided into six aliquots (except for the sperm samples which were not divided into aliquots). SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97–201 kb but only ~27–68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data
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