Attempt origin, DNA removal, and you will genome sequencing

videoinMay 7, 2022

Attempt origin, DNA removal, and you will genome sequencing

Here by whole genome sequencing out of 55 honey bees and by design a premier solution recombination chart in the honey-bee, i learned that crossovers try of GC content, nucleotide range, and you will gene density. We in addition to verified the previous tip one genes shown into the worker thoughts possess oddly higher CO cost. Our investigation hold the consider that diversification out-of staff decisions, however immune setting, was a motorist of the higher crossing-over price during the bees. We discover zero research your crossing-more than rates was followed by a leading NCO rates.

Tips and you will information

Four territories from honeybees (Apis mellifera ligustica Spin) was gathered of an effective bee ranch when you look at the Zhenjiang, China. For every nest consisted of you to definitely king, all those drones, and hundreds of workers. Bees out of three territories was selected to own entire genome sequencing.

The new DNA of every personal is actually removed playing with phenol/chloroform/isoamyl alcohol means. To attenuate the risk of bacterial toxic contamination, brand new stomachs off bees was basically got rid of just before DNA removal. Regarding step three ?g off DNA of per test were used to possess whole genome resequencing as the leftover DNA was remaining having PCR and you may Sanger sequencing. Framework of your own DNA libraries and Illumina sequencing was basically performed on BGI-Shenzhen. Into the short-term, paired-end sequencing libraries which have submit measurements of five hundred bp was in fact created for each decide to try according to maker’s guidelines. Following dos ? 100 bp paired-prevent reads was in fact generated toward IlluminaHiSEq 2000. New queens was sequenced within up to 67? exposure typically, drones on everything thirty five? coverage, and workers in the around 30? visibility (Desk S1 inside Even more document dos). The latest sequences was indeed deposited on GenBank database (accession no. SRP043350).

SNP contacting and you will marker character

Honeybee site genome is installed of NCBI . The latest sequencing checks out was indeed earliest mapped to resource genome which have bwa then realigned which have stampy . Following local realignment as much as indels is did because of the Genome Analysis Toolkit (GATK) , and you will alternatives had been titled by GATK UnifiedGenotyper.

Because of the lower reliability off calling indel versions, only recognized SNPs are used because the indicators. Basic, 920,528 in order to 960,246 hetSNPs was in fact called from inside the for every single queen (Dining table S2 during the Even more file 2). Next, around twenty-two% of those was basically got rid of because the web sites also are hetSNPs from inside the one or more haploid drone (this could echo non-allelic series alignments because of CNVs, sequencing error, or low sequencing quality). Comparable dimensions of the newest hetSNPs along with were seen in people jizz sequencing . Eventually, 671,690 to 740,763 legitimate hetSNPs in the for each nest were utilized while the markers in order to find recombination occurrences (Dining table S2 inside Extra 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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