journal club: single sperm sequencing

Probing Meiotic recombination and aneuploidy single sperm cells (Xie lab)

haplotyping

• homologous recombination, major source of genetic diversity
• determine haplogenotype of each cell. Sequence diploid, not sure which SNPs are on which homologous copy (i.e. which are linked)
• HAP map project, infer linkage genetically by looking at parental

Quake approach

• microfludic device to isolate individual sperm cells.
• lyse, amplify etc.
• MDA — multiple displacement amplification. random primers, polymerize, debranch, new primers anneal.
• use phi29 polymerase, can debranch strands to allow more synthesis
• some sequences amplify more readily than others — amplification bias.
• 30 ~ 50% genome coverage
• ~22 crossovers or ~1 per chromosome.
• some hotspots from bulk data not present in
• No one haplotype over or under-represented (no ‘meiotic drive’)
• Find some ploidy differences

Sunny lab approach

• isolate individual sperm by mouth pipitte
• MALBAC
  1. anneal with random primers.
  2. anything that fully extends forms loops. won’t act as template for new reactions.
  3. Then do PCR after 5 rounds of MALBAC
• MALBAC still some sequence bias, but it’s linear rather than exponetial. substantially better than MDA. Not as good as bulk, (which is still not uniform).
• compare to phased genome and ID crossover events. 95% consistent with family trio.
• 26 crossovers per sperm cell or ~1 per chromosome.
• due to higher coverage, need less contiguous sequence to assign crossover than Quake approach.
• Near TSS, recombination rate is substantially reduced.
• combining to make bulk data of recombination frequency cM/Mb Fit “deCODE” bulk data much better than HapMap.
• find some ploidy differences (aneuploidy – reduced male fertility) (polyploidy by disagreeing sequences).