Truseq pcr

DNA was extracted using https://www.protocols.io/view/seed-sterilization-and-tissue-disruption-for-optim-bpsemnbe. All sequencing libraries where generated using the Illumina TruSeq PCR-free reaction. Accession libraries were sequenced on different instruments: 24 libraries on Illumina HiSeq2500 in paired-end mode with 101 cycles, 70 libraries on Illumina HiSeqX in paired-end mode with 151 cycles, and 72 libraries on Illumina HiSeq3000 in paired-end mode with 151 cycles. Raw Illumina reads and bbtools khist.sh (jgi.doe.gov/data-and-tools/bbtools/) were used to assess the single-copy k-mer count for each accession, based on the main peak in the resultant kmer spectrum (S1 Fig). Haploid genome size was also estimated as part of this analysis (S1 File).

An Illumina TruSeq PCR-free DNA library with ~450 bp insert size of a brown French Bulldog was prepared. We collected 149 million 2 × 125 bp paired-end reads or 14× coverage on a HiSeq2500 instrument (Illumina, San Diego, CA, USA). The reads were mapped to the dog reference genome assembly CanFam3.1 and aligned as described [20 (link)]. Briefly, after trimming adaptor sequences and low-quality bases at the ends of reads with FASTP [21 (link)], BWA version 0.7.13 [22 (link)] was used for the alignment to the canine reference genome. Samtools version 0.1.18 [23 (link)] was used to sort the aligned reads by coordinates, and to produce bam-files. Duplicates were marked with Picard tools [24 ]. The sequence data were submitted to the European Nucleotide Archive with the study accession PRJEB16012 and sample accession SAMEA4504835.

An Illumina TruSeq PCR-free library with ~400 bp insert size of an affected dog from family 1 was prepared. We collected 221 million 2 × 150 bp paired-end reads on a NovaSeq 6000 instrument (35.5 × coverage). Mapping and alignment were performed as described in [15 (link)]. The sequence data were deposited under study accession PRJEB16012 and sample accession SAMEA112638879 at the European Nucleotide Archive.

Genomic DNA was extracted from EDTA blood using the Maxwell RSC Whole Blood DNA kit in combination with the Maxwell RSC instrument (Promega, Dübendorf, Switzerland). An Illumina TruSeq PCR-free library with ~ 420 bp insert size was prepared from case 1. We collected 278 million 2 × 150 bp paired end reads on a NovaSeq 6000 instrument (31.7 × coverage). Mapping to the UU_Cfam_GSD_1.0 reference genome assembly was performed as described^{9 (link)}. The sequence data were deposited under study accession PRJEB16012 and sample accession SAMEA10833663 at the European Nucleotide Archive.

We extracted DNA from the F₂ hybrid offspring using a standard phenol–chloroform protocol. Some individuals that died during development and eggs were extracted in pools of 2–21 individuals, due to low total DNA content in e.g. dead embryos. We measured the DNA content of each extracted sample using Qubit, and pooled samples to get equimolar concentrations of each respective individual. For the intercross, five different pools of F₂ individuals were sequenced: dead embryos (n = 298), eggs (n = 73), dead larvae + dead pupae (n = 72), adult males (n = 76) and adult females (n = 80). The egg pool for the F₂ intercross as well as eggs from the F₂ backcross were sampled three days after laying. Pools were prepared for sequencing using the Illumina TruSeq PCR-free library preparation method and whole-genome re-sequenced (2 × 151 bp paired-end reads with 350 bp insert size) on a single Illumina NovaSeq6000 (S4 flowcell) lane at NGI, SciLifeLab, Stockholm (Table S2).

An Illumina TruSeq PCR-free DNA library with 500 bp insert size of an affected Rough Collie (CL013) was prepared. We collected 227 million 2 × 150 bp paired-end reads on a NovaSeq 6000 instrument (26.9× coverage). Mapping and alignment were performed as described [12 (link)]. The sequence data were deposited under study accession PRJEB16012 and sample accession SAMEA4867926 at the European Nucleotide Archive.

An Illumina TruSeq PCR-free library with an insert size of 350 bp was prepared from one affected dog (GS104). The library was sequenced at 32x genome coverage using 2 x 150 bp reads on an Illumina HiSeq 3000 instrument. Single nucleotide and small indel variants with respect to the CanFam3.1 canine reference genome assembly were called as described [21 (link)]. Variants private to GS104 were identified by filtering variants that were contained in 3 wolf and 188 control dog genomes sequenced for previous projects (S4 Table). Private variants were prioritized according to their predicted impact using SNPeff [22 (link)] and the NCBI annotation release 104.