Sample supply, DNA extraction, and you may genome sequencing

videoinMay 8, 2022

Sample supply, DNA extraction, and you may genome sequencing

Here by whole genome sequencing from 55 honey bees by constructing a premier resolution recombination map in the honey-bee, i found that crossovers is actually regarding the GC posts, nucleotide range, and you can gene density. We also confirmed the former tip that genetics shown during the personnel heads enjoys oddly higher CO prices. All of our studies keep the examine you to definitely diversification out-of employee behavior, although not protected form, was a driver of your highest crossing-more than rates in the bees. We find zero proof that crossing-more rates is with a top NCO rates.

Tips and you will information

Four colonies off honeybees (Apis mellifera ligustica Twist) were collected regarding a beneficial bee farm from inside the Zhenjiang, China. For every single colony consisted of one to king, all those drones, and hundreds of professionals. Bees from about three territories were picked getting whole genome sequencing.

The newest DNA of any private try extracted using phenol/chloroform/isoamyl alcoholic beverages strategy. To attenuate the risk of bacterial contaminants, the fresh new abdomens out of bees was got rid of in advance of DNA removal. Regarding the step 3 ?g off DNA off each shot were used to possess whole genome resequencing given that leftover DNA try remaining for PCR and you may Sanger sequencing. Framework of one’s DNA libraries and you can Illumina sequencing was performed within BGI-Shenzhen. In the short term, paired-avoid sequencing libraries with type size of five-hundred bp was built each take to with respect to the manufacturer’s tips. Then 2 ? one hundred bp matched up-prevent reads have been produced towards the IlluminaHiSEq 2000. The fresh new queens was indeed sequenced within around 67? visibility normally, drones on up to 35? exposure, and you can specialists during the as much as 30? exposure (Desk S1 during the More document 2). The latest sequences had been transferred about GenBank database (accession zero. SRP043350).

SNP contacting and you will marker identification

Honeybee source genome is actually installed off NCBI . The new sequencing checks out had been basic mapped on to resource genome which have bwa immediately after which realigned with stampy . Following local realignment doing indels was did by the Genome Analysis Toolkit (GATK) , and you will versions was entitled by the GATK UnifiedGenotyper.

As a result of the all the way down accuracy of getting in touch with indel variations, only identified SNPs are utilized because the indicators. Basic, 920,528 so you’re able to 960,246 hetSNPs had been called in the for every single queen (Table S2 for the More file 2). Up coming, whenever twenty two% of these was in fact got rid of because web sites are hetSNPs when you look at the one or more haploid drone (this might mirror low-allelic succession alignments caused by CNVs, sequencing mistake, or low sequencing high quality). Comparable dimensions of the brand new hetSNPs and was noticed in individual spunk sequencing . Ultimately, 671,690 to 740,763 credible hetSNPs when you look at the each nest were utilized due to the fact markers so you’re able to locate recombination events (Table S2 when you look at the Most 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.


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