Attempt supply, DNA removal, and you can genome sequencing

May 7, 2022 0 Comments by

Attempt supply, DNA removal, and you can genome sequencing

Here by entire genome sequencing off 55 honey bees and also by developing a top quality recombination map when you look at the honey-bee, i discovered that crossovers are for the GC posts, nucleotide range, and you will gene occurrence. I and additionally confirmed the former idea that genes indicated for the employee heads provides oddly highest CO pricing. Our very own data support the look at one diversity regarding staff member choices, although not resistant setting, is a drivers of one’s higher crossing-over speed during the bees. We find no evidence the crossing-more speed try accompanied by a premier NCO price.

Tips and product

Five territories regarding honeybees (Apis mellifera ligustica Spin) was basically obtained of a great bee ranch when you look at the Zhenjiang, Asia. For each nest consisted of you to definitely king, all those drones, and you may a huge selection of experts. Bees out-of about three territories was in fact chosen to own whole genome sequencing.

The newest DNA of each and every personal is removed having fun with phenol/chloroform/isoamyl alcoholic drinks method. To reduce the risk of bacterial toxic contamination, the latest stomachs from bees had been removed just before DNA extraction. Regarding the step three ?g from DNA out-of for each and every try were utilized to own entire genome resequencing just like the left DNA was remaining to have PCR and you will Sanger sequencing. Construction of your DNA libraries and you can Illumina sequencing was in fact performed at BGI-Shenzhen. Into the short-term, paired-prevent sequencing libraries which have insert measurements of five hundred bp was in fact developed for every test with respect to the manufacturer’s guidelines. Upcoming dos ? one hundred bp matched up-stop checks out was indeed made into the IlluminaHiSEq 2000. The fresh queens had been sequenced during the everything 67? publicity on average, drones in the everything 35? coverage, and you may gurus during the around 29? exposure (Table S1 from inside the Even more file 2). This new sequences was in fact transferred about GenBank databases (accession no. SRP043350).

SNP calling and you will marker personality

Honeybee source genome are installed off NCBI . The fresh new sequencing reads was in fact first mapped to site genome which have bwa after which realigned having stampy . Up coming local realignment as much as indels is actually performed of the Genome Analysis Toolkit (GATK) , and you can variants were titled by GATK UnifiedGenotyper.

Considering the lower accuracy regarding getting in touch with indel alternatives, simply recognized SNPs can be used given that indicators. First, 920,528 so you’re able to 960,246 hetSNPs was indeed named inside for each king (Table S2 countrymatch phone number for the Additional document dos). After that, approximately twenty-two% ones was in fact got rid of because the web sites are also hetSNPs in one haploid drone (this may reflect low-allelic succession alignments due to CNVs, sequencing mistake, otherwise low sequencing quality). Equivalent size of the fresh hetSNPs in addition to was in fact found in person spunk sequencing . Ultimately, 671,690 so you can 740,763 reputable hetSNPs for the each colony were used while the markers to choose recombination occurrences (Table S2 in the A lot 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.