Take to origin, DNA removal, and genome sequencing

Written by on April 21, 2022

Take to origin, DNA removal, and genome sequencing

Right here of the whole genome sequencing off 55 honey bees and by building a leading solution recombination chart in the honey-bee, we discovered that crossovers is regarding the GC content, nucleotide diversity, and you may gene occurrence. I including verified the former suggestion one to genes expressed in employee thoughts has actually surprisingly high CO cost. All of our data hold the see one to variation regarding personnel decisions, not protected form, are a motorist of the higher crossing-more than rates from inside the bees. We discover no proof that crossing-more than rate was followed closely by a high NCO rates.

Tips and you can product

Four territories away from honeybees (Apis mellifera ligustica Twist) was obtained out of an effective bee ranch in the Zhenjiang, China. For every nest contained one to king, all those drones, and you may hundreds of pros. Bees out-of around three colonies was selected to own whole genome sequencing.

The latest DNA each and every individual is actually removed using phenol/chloroform/isoamyl alcohol means. To attenuate the risk of microbial contamination, brand new stomachs out of bees was removed prior to DNA extraction. About step three ?g of DNA away from for each decide to try were used to own entire genome resequencing while the leftover DNA is left having PCR and you can Sanger sequencing. Framework of DNA libraries and Illumina sequencing was in fact performed during the BGI-Shenzhen. Inside brief, paired-end sequencing libraries which have insert sized five hundred bp were constructed for each attempt with respect to the manufacturer’s directions. After that 2 ? 100 bp paired-avoid reads were produced toward IlluminaHiSEq 2000. The latest queens had been sequenced at around 67? visibility typically, drones in the around 35? publicity, and pros during the as much as 30? visibility (Table S1 inside the A lot more document dos). The brand new sequences was in fact deposited on the GenBank database (accession zero. SRP043350).

SNP contacting and you may marker identity

Honeybee reference genome is actually downloaded out of NCBI . Brand new sequencing reads was basically first mapped onto site genome with bwa following realigned having stampy . After that regional realignment up to indels try performed from the Genome Analysis Toolkit (GATK) , and you will alternatives was in fact called from the GATK UnifiedGenotyper.

Due to the lower precision away from calling indel variants, only identified SNPs are utilized just like the indicators. Earliest, 920,528 to help you 960,246 hetSNPs were named in the each king (Dining table S2 within the A lot more file 2). After that, whenever 22% ones was indeed removed because the websites are also hetSNPs for the one or more haploid drone (this could mirror non-allelic series alignments caused by CNVs, sequencing error, otherwise low sequencing top quality). Comparable proportions of the latest hetSNPs also was in fact observed in individual sperm sequencing . Fundamentally, 671,690 so you’re able to 740,763 reputable hetSNPs within the for Phoenix local hookup each colony were utilized because markers to help you detect recombination situations (Desk S2 inside the More file 2).

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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