Decide to try resource, DNA removal, and you can genome sequencing
Right here by the entire genome sequencing away from 55 honey bees and by creating a high resolution recombination chart during the honey bee, i learned that crossovers is actually regarding the GC articles, nucleotide assortment, and gene density. We plus confirmed the previous idea that genetics expressed in personnel thoughts has strangely high CO pricing. The study contain the glance at one to diversification out of worker choices, however resistant function, is a driver of your own higher crossing-more speed for the bees. We find zero evidence the crossing-over rates try followed by a premier NCO price.
Steps and you will content
Five territories regarding honeybees (Apis mellifera ligustica Spin) had been amassed of a good bee farm inside the Zhenjiang, Asia. For each colony contained you to definitely king, dozens of drones, and you will a huge selection of experts. Bees off about three colonies were picked for whole genome sequencing.
The DNA each and every individual are extracted playing with phenol/chloroform/isoamyl alcoholic beverages approach. To minimize the risk of bacterial pollution, the new stomachs of bees were removed before DNA extraction. Throughout the 3 ?g from DNA from for every single attempt were utilized to have whole genome resequencing as the remaining DNA try leftover having PCR and you will Sanger sequencing. Framework of your DNA libraries and you will Illumina sequencing was basically performed within BGI-Shenzhen. From inside the short term, paired-stop sequencing libraries with input size of 500 bp was indeed built for every single take to according to maker’s tips. Up coming 2 ? 100 bp coordinated-avoid checks out were made for the IlluminaHiSEq 2000. This new queens was basically sequenced in the just as much as 67? coverage an average of, drones from the everything thirty-five? visibility, and you will workers in the as much as 29? publicity (Table S1 in the More file dos). New sequences was in fact transferred regarding the GenBank databases (accession zero. SRP043350).
SNP getting in touch with and marker identity
Honeybee source genome try installed away from NCBI . The fresh new sequencing reads were earliest mapped to resource genome with bwa and then realigned that have stampy . Next regional realignment around indels is actually performed by the Genome Investigation Toolkit (GATK) , and you will versions were named of the GATK UnifiedGenotyper.
As a result of the all the way down reliability out of calling indel alternatives, only understood SNPs can be used as the indicators. Very first, 920,528 so you’re able to 960,246 hetSNPs was basically entitled within the for each and every queen (Dining table S2 for the Additional document 2). Next, whenever twenty-two% of these was eliminated because those sites are also hetSNPs into the one or more haploid drone (this could reflect non-allelic sequence alignments because of CNVs, sequencing error, otherwise low sequencing high quality). Equivalent size of cybermen the brand new hetSNPs together with was indeed noticed in human spunk sequencing . In the end, 671,690 to 740,763 reputable hetSNPs in for every colony were utilized because the markers to help you position recombination occurrences (Table S2 when you look at the Extra document 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.
