Sample provider, DNA removal, and genome sequencing
Right here by entire genome sequencing regarding 55 honey bees and by developing a top solution recombination map for the honey bee, i learned that crossovers is associated with GC posts, nucleotide range, and you can gene thickness. We including verified the previous idea you to genetics expressed when you look at the personnel minds have oddly highest CO costs. Our very own investigation keep the check one diversity away from staff conclusion, but not protected form, is actually a motorist of your own large crossing-over rates during the bees. We discover no evidence that the crossing-more price are with a top NCO price.
Steps and you can product
Four colonies out of honeybees (Apis mellifera ligustica Spin) were built-up of a beneficial bee farm within the Zhenjiang, Asia. Per nest contains you to king, all those drones, and you can numerous professionals. Bees out-of three colonies was in fact picked to have whole genome sequencing.
The new DNA each and every personal was removed using phenol/chloroform/isoamyl alcohol approach. To minimize the risk of bacterial contaminants, the fresh abdomens of bees was removed before DNA extraction. About step 3 ?g of DNA regarding for each shot were used getting entire genome resequencing as left DNA is actually left to have PCR and Sanger sequencing. Framework of your DNA libraries and you will Illumina sequencing were did at BGI-Shenzhen. Into the temporary, paired-prevent sequencing libraries which have submit sized 500 bp were created per take to with regards to the maker’s instructions. Upcoming 2 ? one hundred bp matched-end reads was indeed made into the IlluminaHiSEq 2000. The brand new queens had been sequenced from the just as much as 67? coverage normally, drones during the whenever thirty-five? visibility, and professionals on just as much as 29? publicity (Table S1 from inside the Even more file dos). The new sequences was in fact deposited throughout the GenBank databases (accession zero. SRP043350).
SNP contacting and you can marker character
Honeybee resource genome are downloaded out-of NCBI . The sequencing reads was basic mapped to site genome having bwa immediately after which realigned that have stampy . Up coming regional realignment up to indels was did from the Genome Data Toolkit (GATK) , and you can variations were named because of the GATK UnifiedGenotyper.
Because of the down accuracy off getting in touch with indel versions, merely identified SNPs are utilized given that markers. Earliest, 920,528 so you can 960,246 hetSNPs was basically titled in clover dating profiles the for every king (Table S2 during the A lot more file dos). Following, approximately twenty two% of those have been eliminated because the websites are also hetSNPs for the one or more haploid drone (this may reflect non-allelic succession alignments as a result of CNVs, sequencing error, otherwise low sequencing quality). Equivalent proportions of the latest hetSNPs including have been present in peoples spunk sequencing . In the end, 671,690 so you can 740,763 reputable hetSNPs when you look at the for each colony were utilized as the markers so you’re able to detect recombination occurrences (Table S2 within 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.
