Decide to try source, DNA removal, and you will genome sequencing

Decide to try source, DNA removal, and you will genome sequencing

Right here by the entire genome sequencing out of 55 honey bees and by creating a leading quality recombination chart in the honey-bee, we learned that crossovers is actually for the GC stuff, nucleotide range, and you may gene thickness. I plus affirmed the previous tip you to genes indicated during the worker thoughts has actually surprisingly higher CO pricing. Our very own investigation keep the evaluate you to diversification off worker decisions, however resistant setting, try a motorist of your highest crossing-more rate in bees. We discover zero evidence the crossing-over speed is followed closely by a high NCO price.

Actions and you can content

Five territories from honeybees (Apis mellifera ligustica Twist) had been compiled off a bee farm inside the Zhenjiang, Asia. Each nest contained one king, those drones, and you will a huge selection of gurus. Bees from three colonies was in fact chose to own entire genome sequencing.

The new DNA of every personal was extracted having fun with phenol/chloroform/isoamyl liquor approach. To attenuate the risk of bacterial contaminants, the newest stomachs from bees was indeed eliminated just before DNA removal. Regarding the step 3 ?g of DNA out-of for each try were utilized to possess entire genome resequencing due to the fact remaining DNA try left to possess PCR and Sanger sequencing. Framework of one’s DNA libraries and you will Illumina sequencing was performed from the BGI-Shenzhen. Inside short term, paired-prevent sequencing libraries having insert measurements of five hundred bp was built each decide to try with respect to the manufacturer’s instructions. Upcoming 2 ? one hundred bp matched-avoid reads were generated towards IlluminaHiSEq 2000. The brand new queens was in fact sequenced within up to 67? publicity an average of, drones within approximately thirty five? exposure, and you can professionals at just as much as 30? visibility (Desk S1 inside More document 2). New sequences were placed in the GenBank database (accession zero. SRP043350).

SNP contacting and you can marker personality

Honeybee resource genome is installed from NCBI . New sequencing reads have been very first mapped to reference genome that have bwa then realigned with stampy . Following regional realignment up to indels is did by the Genome Study Toolkit (GATK) , and you will alternatives were titled by GATK UnifiedGenotyper.

Due to the straight down reliability out of getting in touch with indel alternatives, simply recognized SNPs are used just like the markers. Earliest, 920,528 to help you 960,246 hetSNPs was in fact titled inside each king (Desk S2 for the A lot more file 2). After that, as much as 22% of those had been got rid of due to the fact that web sites are hetSNPs for the a minumum of one haploid drone (this could echo low-allelic sequence alignments as a result of CNVs, sequencing error, otherwise reduced sequencing quality). Similar proportions of this new hetSNPs plus was found in human sperm sequencing . Eventually, 671,690 to help you 740,763 legitimate hetSNPs inside the for every nest were utilized as the markers so you can discover recombination situations (Table S2 in Additional 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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