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Test resource, DNA extraction, and genome sequencing

Test resource, DNA extraction, and genome sequencing

Right here by whole genome sequencing off 55 honey bees and also by design a leading resolution recombination chart in the honey bee, i discovered that crossovers are of this GC articles, nucleotide diversity, and you may gene thickness. We plus affirmed the former idea that genetics expressed within the worker minds enjoys unusually high CO costs. All of our research hold the consider one to diversification out of employee decisions, not resistant means, are a driver of the highest crossing-more than price inside the bees. We discover no facts that crossing-more than speed is followed closely by a premier NCO speed.

Methods and you will product

Five colonies out of honeybees (Apis mellifera ligustica Spin) was amassed from a great bee farm for the Zhenjiang, Asia. For each and every colony consisted of that king, dozens of drones, and you will a huge selection of workers. Bees of about three colonies was indeed selected to possess whole genome sequencing.

The newest DNA of each and every individual is actually extracted having fun with phenol/chloroform/isoamyl liquor strategy. To reduce the risk of bacterial contaminants, new stomachs out of bees were got rid of ahead of DNA extraction. In the 3 ?g from DNA out-of each test were used to own whole genome resequencing just like the left DNA is remaining getting PCR and you will Sanger sequencing. Build of DNA libraries and you can Illumina sequencing were performed on BGI-Shenzhen. From inside the temporary, paired-end sequencing libraries having insert measurements of five-hundred bp have been created for each attempt with respect to the manufacturer’s recommendations. Up coming dos ? 100 bp coordinated-prevent reads was in fact made toward IlluminaHiSEq 2000. The latest queens was in fact sequenced during the approximately 67? exposure normally, drones in the as much as 35? exposure, and you can experts during the everything 30? coverage (Desk S1 inside the More document 2). New sequences was indeed transferred on GenBank databases (accession zero. SRP043350).

SNP getting in touch with and you will marker identification

Honeybee reference genome is actually installed off NCBI . The sequencing reads was in fact first mapped onto resource genome that have bwa right after which realigned with stampy . Next regional realignment doing indels is did because of the Genome Research Toolkit (GATK) , and you will alternatives was basically called of the GATK UnifiedGenotyper.

As a result of the all the way down accuracy out-of contacting indel versions, simply identified SNPs are utilized as indicators. Earliest, 920,528 so you’re able to 960,246 hetSNPs was in fact named into the for every king (Table S2 from inside the Most file 2). Then, whenever 22% of these were eliminated because these sites are also hetSNPs inside the one or more haploid drone (this might reflect low-allelic succession alignments due to CNVs, sequencing mistake, or lower sequencing high quality). Similar dimensions of the brand new hetSNPs and have been present in person cum sequencing . Finally, 671,690 in order to 740,763 reputable hetSNPs in the per colony were utilized as the markers so you can discover recombination occurrences (Desk S2 inside the A lot 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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