Sample resource, DNA extraction, and genome sequencing

Sample resource, DNA extraction, and genome sequencing

Right here by the entire genome sequencing regarding 55 honey bees and also by developing a high quality recombination map in honey bee, i found that crossovers try of the GC articles, nucleotide variety, and you will gene density. We as well as affirmed the former idea one to genetics expressed within the employee heads enjoys strangely large CO cost. All of our analysis hold the evaluate you to diversity off staff member behavior, but not resistant form, are a drivers of large crossing-over rate for the bees. We find zero facts your crossing-more rates is actually with a top NCO speed.

Methods and you can information

Four territories regarding honeybees (Apis mellifera ligustica Twist) had been compiled away from an effective bee ranch within the Zhenjiang, Asia. Each colony contained one queen, all those drones, and you will a huge selection of pros. Bees off about three colonies was indeed chosen for entire genome sequencing.

The DNA each and every individual is extracted playing with phenol/chloroform/isoamyl alcoholic beverages approach. To attenuate the possibility of bacterial contaminants, the fresh new abdomens regarding bees were got rid of in advance of DNA removal. Regarding step three ?g of DNA out of each sample were used to own entire genome resequencing since the leftover DNA was kept getting PCR and you can Sanger sequencing. Framework of DNA libraries and you may Illumina sequencing had been performed on BGI-Shenzhen. In the short term, paired-avoid sequencing libraries with type measurements of five hundred bp was indeed constructed each shot depending on the maker’s rules. Next dos ? a hundred bp paired-avoid checks out have been generated on IlluminaHiSEq 2000. New queens was indeed sequenced during the approximately 67? publicity on average, drones within approximately thirty-five? visibility, and pros within everything 29? exposure (Dining table S1 in the Even more document dos). The latest sequences was in fact deposited from the GenBank databases (accession no. SRP043350).

SNP contacting and you can marker identification

Honeybee source genome try downloaded regarding NCBI . Brand new sequencing checks out have been first mapped to source genome that have bwa then realigned having stampy . Next local realignment up to indels is actually performed because of the Genome Study Toolkit (GATK) , and alternatives was in fact titled from the GATK UnifiedGenotyper.

Because of the down accuracy away from getting in touch with indel versions, merely identified SNPs are utilized because the markers. Very first, 920,528 so you’re able to 960,246 hetSNPs was in fact named in per queen (Dining table S2 for the More document 2). Up coming, up to twenty-two% of those had been got rid of because those sites are hetSNPs inside the a minumum of one haploid drone (this may echo non-allelic series alignments due to CNVs, sequencing error, otherwise reduced sequencing top quality). Comparable dimensions of the hetSNPs in addition to was basically present in people sperm sequencing . Eventually, 671,690 so you’re able to 740,763 legitimate hetSNPs inside per nest were utilized as the indicators so you’re able to position recombination events (Dining table S2 in the Extra file 2).

Haploid phasing

For each colony, the identified markers were used for haploid phasing. The linkage of every two adjacent markers was inferred to determine the two chromosome haplotypes of the queen by comparing the SNP linkage information across all drones from the same colony. Detailed methods were described in Lu’s study . In brief, for each pair of adjacent hetSNPs, for example A/G and C/T, there could be two types of link in the queen ‘A-C, G-T’ or ‘A-T, G-C’. Assuming fdating app recombination events are low probability, if more ‘A-C, G-T’ drones are found than ‘A-T, G-C’ drones, then ‘A-C, G-T’ is assumed to be the correct link in the queen and vice versa. The two haplotypes can be clearly discriminated between >99% of ple). For linkage of the <1% markers, as shown in Additional file 1: Figure S2B, between markers at ‘LG1:20555174' and ‘LG1:20555456' , there are 14 ‘A-A or G-G' type drones against 1 ‘A-G or G-A' type drone, so ‘A-A, G-G' is assumed to be the correct link in queen and a recombination event is identified at this site in sample I-9.

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