Decide to try provider, DNA removal, and genome sequencing

Here because of the entire genome sequencing regarding 55 honey bees by developing a premier quality recombination chart within the honey bee, we discovered that crossovers try associated with the GC articles, nucleotide range, and you may gene density. I together with confirmed the previous tip that family genes conveyed in personnel minds enjoys strangely higher CO rates. All of our study support the glance at one to diversification from staff conclusion, yet not resistant setting, is a motorist of one’s highest crossing-more than rate in bees. We find no evidence that crossing-more than speed try followed by a leading NCO rate.

Procedures and you may information

Five territories out of honeybees (Apis mellifera ligustica Twist) was in fact compiled from a great bee farm within the Zhenjiang, China. For every nest contains one king, those drones, and you may a huge selection of specialists. Bees away from three colonies have been chose getting whole genome sequencing.

Brand new DNA each and every personal is actually extracted using phenol/chloroform/isoamyl alcoholic beverages strategy. To reduce the possibility of bacterial contamination, the stomachs of bees was basically removed ahead of DNA extraction. On step three ?g regarding DNA out of per sample were utilized for entire genome resequencing as the remaining DNA try left having PCR and you will Sanger sequencing. Construction of one’s DNA libraries and you may Illumina sequencing was indeed did in the BGI-Shenzhen. For the brief, paired-end sequencing libraries which have type sized five-hundred bp was constructed for each and every take to according to manufacturer’s recommendations. Upcoming dos ? a hundred bp coordinated-prevent reads was basically produced into the IlluminaHiSEq 2000. New queens had been sequenced during the around 67? publicity an average of, drones in the whenever 35? publicity, and you can experts during the around 31? coverage (Dining table S1 into the Most document 2). This new sequences was basically transferred regarding GenBank databases (accession zero hookup numbers near me Thunder Bay. SRP043350).

SNP contacting and you can marker identity

Honeybee source genome try downloaded of NCBI . The latest sequencing reads have been first mapped onto site genome with bwa after which realigned with stampy . Up coming regional realignment up to indels is actually performed by the Genome Data Toolkit (GATK) , and versions was in fact called by GATK UnifiedGenotyper.

Because of the straight down reliability regarding calling indel alternatives, only understood SNPs are utilized due to the fact indicators. First, 920,528 so you’re able to 960,246 hetSNPs was in fact entitled inside the per queen (Desk S2 inside the Additional file dos). Following, approximately twenty two% of them had been removed due to the fact that those sites are hetSNPs into the one haploid drone (this could echo low-allelic sequence alignments considering CNVs, sequencing mistake, otherwise lowest sequencing quality). Equivalent proportions of the new hetSNPs in addition to had been noticed in people sperm sequencing . In the end, 671,690 to help you 740,763 credible hetSNPs inside each nest were utilized because the markers in order to choose recombination events (Table S2 for the Even more 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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