Truseq pcr free kit

For each treatment, three samples were selected for shotgun metagenomics, resulting in a total of 18 sequenced samples. Libraries were prepared with a TruSeq PCR-free kit (Illumina). Samples were sequenced on a NovaSeq 6000 with a 2 × 151 setup, resulting in 800 million pair-reads. Low quality reads and adapters were removed with Trimmomatic v0.39^{70 (link)}. Co-assembly of short reads was done with Megahit^{71 (link)} using the --presets meta-large setting, resulting in 1,975,712 contigs of ≥1 kb, and N50 of 4375 bp. Reads were mapped to the co-assembly using Bowtie2 v2.3.5^{72 (link)}. Protein coding sequences in all the assembled contigs were identified with Prodigal v.2.6.3^{73 (link)}, using the meta prediction mode.

The differentiation and structural variation studies were based on resequencing of four pools of DNA: two pools of male or female progenies from the two biparental populations 74F × Kabusa (36 females and 38 males) and 74F × 14 M (32 females and 46 males). Each pool consisted of eight leaf punches per flowering progeny to balance the DNA quantity and by choosing leaves close to flowers to avoid vine mixing. DNA extraction and quality checks were carried out as previously described at the GenoAgap platform (CIRAD, Montpellier, France). Library preparation and sequencing was conducted using the Illumina TruSeq PCR-Free kit. Paired-end (2 × 150 bp) sequencing was conducted on an Illumina HiSeq3000 system. Both library preparation and sequencing were performed at the GeT-PlaGe platform (INRAe, Castanet-Tolosan, France).

Genomic DNA isolated from the cecal contents of 5 mice per treatment group was used for metagenomic analysis. Libraries were prepared using the Illumina TruSeq PCR-free kit following vendor protocols and sequenced at the University of Wisconsin Biotechnology Center’s DNA Sequencing Facility. All samples were run on a single NovaSeq6000 2 × 150 S4-Flowcell lane. The resulting sequences were trimmed for quality using Trimmomatic (version 0–39) and then aligned against reference host genomes (Mus musculus GRCm38_Rel98) with bowtie2 (version 2.3.4) to remove host reads (average host alignment rate was 5.3%) leaving only high-quality, non-host reads. Cleaning ultimately resulted in an average of 29.4 million paired-end reads per sample.

Genomic DNA of eight representative trees of the SU collection and one of the parental trees of the mapping population, ‘Yama-Zakura’, were digested with NEBNext dsDNA Fragmentase (New England BioLabs, Ipswich, MA, USA) for whole genome shotgun library preparation using the Illumina TruSeq PCR-free Kit. The sequences were determined on the Illumina NextSeq platform. Read trimming, read mapping to the CYE_r3.1 sequence and SNP identification were performed as described above. Effects of SNPs on gene functions were evaluated using SnpEff v. 4.2.⁶¹ (link)

One hundred Nile tilapia broodstock samples from the 15^th generation of the core GIFT Nile tilapia-breeding nucleus of WorldFish at the Aquaculture Extension Center in Jitra (Kedah, Malaysia) were used for DNA sequencing for SNP discovery. Caudal fin clips were sampled and preserved in absolute ethanol at -20° until shipment from Malaysia to The Roslin Institute (University of Edinburgh, UK) for DNA extraction, sequencing and genetic analysis.
Genomic DNA was isolated from the tilapia fin clips using a salt-based extraction method (Aljanabi and Martinez 1997 (link)). The integrity of the DNA samples was assessed by performing an agarose gel electrophoresis. DNA quality was also evaluated by estimating the 260/280 and 260/230 ratios on a NanoDrop 1000 UV spectrophotometer. The concentration of the DNA extractions was measured with the Qubit dsDNA BR assay kit (Invitrogen, Life technologies). Samples were diluted to 50 ng/ul and then combined in equimolar concentrations to generate two pools of 50 (different) individuals each. Library preparation and sequencing services were provided by Edinburgh Genomics (University of Edinburgh, UK). DNA pools were prepared for sequencing using a TruSeq PCR-free kit (Illumina, San Diego). The two pools were then sequenced at a minimum 90X depth of coverage on an Illumina HiSeq X platform with a 2x150 bp read length.

Whole-genome sequence data and genotype calls for 121 hESC lines were obtained from Merkle et al.¹⁹ . We denote these as “Merkle_batch1”. Nine additional hESCs were sequenced in another batch from Merkle et al.¹⁹ , denoted as “Merkle_batch2”. We further used whole-genome sequence data from 326 iPSC lines from the HipSci Project^{20 (link)} (ENA accession number: PRJEB15299), denoted as “HipSci”. We sequenced an additional 15 hESCs and 17 iPSCs (dbGaP accession number: phs001957), denoted as “in_house_hESC” and “in_house_iPSC”, respectively.
For the in-house datasets, DNA was extracted using the MasterPure Complete DNA and RNA Purification Kit (Lucigen). Sequencing libraries were prepared using the Illumina TruSeq PCR-free kit and sequenced on an Illumina HiSeq X Ten to ~16-fold coverage with 150 × 2 paired-end reads. Sequencing was performed at GeneWiz (South Plainfield, NJ). Reads were aligned to GRCh37 using BWA, and genetic variants were called following the GATK Best Practices. Variants were filtered using GATK’s variant quality score recalibration, such that SNPs had a 99.9% sensitivity to true variants (HapMap 3.3 and Omni 2.5M)^{58 (link)} and a 99.0% sensitivity to true indels (Mills / 1000 Genomes indels)^{59 (link),60 (link)}.