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Attempt resource, DNA removal, and you will genome sequencing

Here because of the whole genome sequencing regarding 55 honey bees and also by design a premier solution recombination map in honey bee, i learned that crossovers is actually in the GC stuff, nucleotide variety, and gene occurrence. I as well as confirmed the former idea you to genetics indicated during the staff member thoughts has actually surprisingly highest CO cost. Our very own data contain the view you to variation from worker conclusion, but not immune function, is a driver of your high crossing-more rates from inside the bees. We discover no research your crossing-more than price try with a top NCO price.

Measures and you may information

Four territories off honeybees (Apis mellifera ligustica Twist) was basically accumulated regarding a great bee farm into the Zhenjiang, Asia. For each and every colony contained you to definitely queen, all those drones, and a huge selection of pros. Bees out-of about three colonies was indeed picked getting entire genome sequencing.

This new DNA of every personal try removed having fun with phenol/chloroform/isoamyl alcoholic drinks means. To minimize the risk of bacterial pollution, the latest abdomens off bees was in fact eliminated before DNA removal. About 3 ?g out of DNA away from for every single take to were used for whole genome resequencing while the kept DNA are kept to own PCR and you may Sanger sequencing. Structure of your DNA libraries best way to find a hookup in Plymouth and you may Illumina sequencing were performed on BGI-Shenzhen. For the brief, paired-stop sequencing libraries having enter size of five hundred bp was indeed created for each and every sample according to the manufacturer’s information. Upcoming 2 ? a hundred bp paired-avoid reads were produced with the IlluminaHiSEq 2000. The fresh queens was in fact sequenced in the everything 67? visibility normally, drones at up to 35? coverage, and you may specialists on whenever 29? coverage (Desk S1 during the Additional file 2). The fresh sequences had been transferred on GenBank databases (accession zero. SRP043350).

SNP contacting and you can marker personality

Honeybee reference genome was downloaded away from NCBI . The sequencing checks out was indeed very first mapped on to reference genome with bwa then realigned that have stampy . Next local realignment around indels are performed because of the Genome Studies Toolkit (GATK) , and you can alternatives was indeed called of the GATK UnifiedGenotyper.

As a result of the all the way down reliability out of calling indel alternatives, merely known SNPs can be used because the indicators. Very first, 920,528 in order to 960,246 hetSNPs had been called into the for every queen (Dining table S2 inside A lot more file dos). Upcoming, up to twenty two% of them was indeed removed because the websites are hetSNPs in a minumum of one haploid drone (this may reflect non-allelic sequence alignments as a result of CNVs, sequencing mistake, or low sequencing quality). Equivalent dimensions of the brand new hetSNPs as well as have been found in human spunk sequencing . In the end, 671,690 so you’re able to 740,763 credible hetSNPs inside per nest were used once the indicators so you’re able to position recombination events (Table S2 for the Most document 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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