Shot resource, DNA removal, and you may genome sequencing

Shot resource, DNA removal, and you may genome sequencing

Right here from the entire genome sequencing off 55 honey bees by building a high quality recombination map into the honey bee, i unearthed that crossovers are in the GC content, nucleotide variety, and you will gene density. We also affirmed the previous tip one to genetics conveyed from inside the personnel heads features unusually highest CO prices. Our study contain the evaluate you to diversity regarding staff member behavior, but not resistant means, was a drivers of the high crossing-over rates in bees. We find zero research that the crossing-more rate is accompanied by a high NCO rate.

Methods and you will content

Five territories off honeybees (Apis mellifera ligustica Spin) had been obtained away from good bee farm within the Zhenjiang, China. For each and every nest contained one king, those drones, and you may a huge selection of workers. Bees out-of three colonies had been selected having entire genome sequencing.

Brand new DNA of every personal are extracted using phenol/chloroform/isoamyl liquor strategy. To minimize the risk of microbial contamination, this new stomachs out of bees were eliminated in advance of DNA removal. About step three ?g away from DNA of for each sample were used to possess entire genome resequencing since the leftover DNA try left for PCR and Sanger sequencing. Construction of the DNA libraries and you may Illumina sequencing was performed at BGI-Shenzhen. Inside the short-term, paired-avoid sequencing libraries with insert sized five-hundred bp was indeed constructed for every single take to with respect to the maker’s advice. Then 2 ? 100 bp matched-end checks out were produced on the IlluminaHiSEq 2000. The queens was basically sequenced on everything 67? visibility typically, drones during the approximately thirty five? visibility, and gurus in the everything 31? coverage (Dining table S1 inside Extra file 2). The fresh new sequences was deposited regarding the GenBank database (accession zero. SRP043350).

SNP calling and you will marker identity

Honeybee resource genome is actually installed regarding NCBI . The sequencing reads have been basic mapped on to reference genome which have bwa and then realigned with stampy . Following local realignment up to indels is performed of the Genome Study Toolkit (GATK) , and you will variations was in fact collarspace reviews entitled by GATK UnifiedGenotyper.

Due to the lower precision from getting in touch with indel alternatives, simply known SNPs are utilized just like the indicators. First, 920,528 to help you 960,246 hetSNPs was indeed entitled inside the each queen (Table S2 in Extra document dos). Then, around twenty-two% of those was indeed removed because the web sites are also hetSNPs in the a minumum of one haploid drone (this could mirror low-allelic sequence alignments considering CNVs, sequencing error, otherwise reasonable sequencing high quality). Similar dimensions of brand new hetSNPs including was indeed present in human sperm sequencing . Ultimately, 671,690 to help you 740,763 reliable hetSNPs when you look at the per colony were used just like the indicators so you can discover recombination incidents (Desk S2 within the Most file dos).

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.

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