Shot supply, DNA extraction, and you may genome sequencing

Here because of the entire genome sequencing of 55 honey bees and also by creating a premier solution recombination chart from inside the honey-bee, we discovered that crossovers is actually for the GC stuff, nucleotide diversity, and gene thickness. We and additionally confirmed the former idea that family genes conveyed during the personnel brains have oddly high CO rates. The studies contain the see one to variation of personnel choices, although not immune function, try a drivers of your own higher crossing-more than price from inside the bees. We discover zero research your crossing-more price is actually with a high NCO price.

Methods and you will material

Four territories out-of honeybees (Apis mellifera ligustica Spin) have been obtained of a bee ranch within the Zhenjiang, Asia. For each nest contains you to definitely king, dozens of drones, and you will countless gurus. Bees off about three colonies was chose to own entire genome sequencing.

The newest DNA of each and every personal was removed using phenol/chloroform/isoamyl alcohol method. To attenuate the risk of bacterial toxic contamination, the new stomachs away from bees have been eliminated before DNA removal. From the step 3 ?g out of DNA out-of per test were used having whole genome resequencing due to the fact leftover DNA try leftover getting PCR and you may Sanger sequencing. Construction of one’s DNA libraries and you can Illumina sequencing were did in the BGI-Shenzhen. During the short-term, paired-stop sequencing libraries having input sized five hundred bp have been created each sample depending on the manufacturer’s instructions. After that dos ? 100 bp coordinated-avoid reads had been generated towards the IlluminaHiSEq 2000. The latest queens have been sequenced within as much as 67? publicity an average of, drones at the just as much as thirty five? exposure, and you may gurus on everything 30? exposure (Dining table S1 inside the Additional file 2). Brand new sequences was deposited on GenBank databases (accession zero. SRP043350).

SNP calling and you will marker personality

Honeybee source genome is actually installed from NCBI . New sequencing reads was basically very first mapped to site genome which have bwa after which realigned that have stampy . Next local realignment around indels was did because of the Genome Analysis Toolkit (GATK) , and alternatives was in fact entitled by the GATK UnifiedGenotyper.

Considering the down precision away from calling indel alternatives, just known SNPs can be used while the markers. First, 920,528 in order to 960,246 hetSNPs had been named inside for each and every queen (Dining table S2 into the Additional document dos). Upcoming, up to 22% ones was basically got rid of because those sites are also hetSNPs in the one haploid drone (this might reflect non-allelic succession alignments for the reason that CNVs, sequencing mistake, or lower sequencing high quality). Equivalent dimensions of the fresh hetSNPs and have been observed in person sperm sequencing . Eventually, 671,690 so you’re able to 740,763 reliable hetSNPs from inside the each nest were utilized while the markers to help you discover recombination occurrences (Table S2 inside the Most 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 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.