Ed DNA at room temperature for 30 min, using, 4 L T4 DNA Ligase (USB/Affymetrix, Santa Clara, CA, USA) in 1?Ligase Buffer (NEB, Frankfort, Germany) and a total volume of 40 L. After an additional purification with 2?volume of AMPure XP beads each sample was quantified using the Quant-itTM ds DNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and a GENios Pro microplate reader (Tecan, Crailsheim, Germany). Samples were subsequently pooled in an equimolar way. The use of one biotinylated and one nonbiotinylated adapter allowed the specific selection of proper P5-P7 library fragments by selective binding to streptavidin-coated beads and subsequent denaturing of the bound double-strands as described [35]. Binding and wash steps to 30 L Dynabeads?MyOne (Thermo Fisher Scientific, Schwerte, Germany) were carried out according to manufacturer’s protocol. P5-P7 strands were eluted by denaturing the double-stranded DNA with alkaline lysis buffer: 400 mM KOH, 10 mM EDTA, 80 mM DTT. Each eluted library pool was diluted 1:10 and 2 L were used as template in the final library PCR containing 100 M of each dNTP, 2 units FastStart Taq DNA Polymerase, 1 L 50xEvaGreen?and 400 M P5-Universal-Primer and 400 M P7-Index-Primer (Additional file 2: Table S1). Finally, 6 pools were size-selected to 200?00 bp long fragments using an E-Gel electrophoresis system (Thermo Fisher Scientific, Waltham, MA, USA) and quantified using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were then sequenced either on the Illumina HiSeq 2000 or the Illumina NextSeq 500 system, yielding reads with maximum single end read length of 100 or 151 bp, respectively (Additional file 6: Table S5).Raw read filtering, processing and population genomic analysisbuilding the catalogue (-n 1). The file catalog.snps.tsv was screened for erroneous SNP alleles resulting from in silico elongation of reads to 95 bp. All loci containing the erroneous `N’-allele were blacklisted using the option in subsequent analyses. Secondly, the population script was applied in order to calculate population genetic parameters. As the present study explicitly aimed to further decipher determinants for temperature-dependent sex reversal, exclusion of autosomal genes or other sex-skewing modifiers was paramount. As only family 1 was devoid of males in the control group, initially this family was chosen for the ddRADseq approach. Subsequently families 2 and 3, which showed some sex-reversal in the Nutlin-3a chiralMedChemExpress Nutlin (3a) controls, too, were additionally sequenced for a case-control approach. Independent runs of the populations script were performed comprising the following data sets: data set 1) comparison of 20 temperature-treated males with 20 females in family 1; 2) comparison of 60 temperaturetreated males (affected cases) and 60 temperaturetreated but non-masculinized females (unaffected controls) from families 1, 2, and 3. All data sets were filtered equally to a stack depth of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27527552 >5 (-m 5), a minor allele frequency of >0.01 ( in_maf 0.01), and a minimum percentage of individuals in the population required to process the locus >70 (-r 0.7), ddRAD tags were requested to be present in all populations within a data set (-p 2). SNP and haplotype-based F-statistics were requested using the stats command, kernel smoothing of FST was enabled using the option applying a default window size of 3 (150 Kbp). The fixation index FST was calculated with Stacks 1.34 [20] using an adapted formula, accounting for.
