Truseq dna pcr free

After TruSeq DNA PCR-free (Illumina Inc., San Diego, CA, USA) library preparation, WGS was performed on genomic DNA isolated from peripheral blood at Baylor College of Medicine (Houston, TX, USA) using Illumina HiSeq instrument to obtain an average of 36-fold genome coverage (range from 25- to 39-fold). Paired-end reads were aligned to human reference genome (GRCh37/hg19) using BWA-mem [43 (link)]. SNVs were called from aligned reads using GATK (v2.7) as previously described [44 (link)–46 (link)].
After applying filters for variant quality (GATK VQSR, ≥10 total reads, genotype quality score ≥30 and alternate allele fraction between 0.2–0.8 and ≥0.9 for heterozygous and homozygous SNPs, respectively), we retained for further analysis those SNVs with MAF ≤1% in the 1000 Genomes Project (1000G) [6 (link)] and Genome Aggregation Database (GnomAD) [7 (link)] (http://gnomad.broadinstitute.org/about) databases, as well as in our cohort (n = 247).

DNA was extracted in triplicate from the same 50 mL sample using the FastDNA ${}^{\circledR }$ Spin Kit for Soil (MP Biomedicals GmbH, Eschwege, Germany) in combination with the Genomic DNA Clean & Concentrator^TM purification kit (Zymo Research, Irvine, USA) according to the manufacturer’s instructions. Metagenomic sequence libraries were constructed in triplicate for each DNA triplicate according to the “TruSeq DNA PCR-free” protocol (Illumina Inc., San Diego, CA, USA) and subsequently sequenced on the Illumina HiSeq platform (2 × 250 bp, paired-end mode, two lanes per sample).

Genomic DNA was extracted from testis and leg muscle samples of the two Spain individuals using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich) following the manufacturer’s indications. Libraries were constructed using the Illumina TruSeq DNA PCR-Free method with an insert size of ~350 bp and sequenced on the HiSeq X Ten platform, yielding at least 17 Gb per sample (coverage ~14×) of 2 × 151 bp paired-end reads. RNA was extracted from testis and leg muscle from the same individuals using the RNeasy Lipid Tissue Kit (Qiagen) following the manufacturer’s indications. Libraries were constructed with the TruSeq mRNA Sample Prep Kit v2 and sequenced using the HiSeq4000 platform, yielding ~10 Gb per sample of 2 × 101 bp paired-end reads. Trimming was done using Trimmomatic^{38 (link)} v0.33 with options ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:100.

Virion DNA was extracted as described by Jakočiūnė and Moodley (72 (link)) after PEG precipitation, starting from step 3.2. Residual bacterial DNA was removed by treating samples with DNase I and RNase at 37°C. The phage protein capsid was digested with Proteinase K, and the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) was used to purify the DNA. The quantity and quality of the purified DNA was checked by a Qubit fluorometer and a NanoDrop spectrometer. Library preparation (Illumina TruSeq DNA PCR-Free), sequencing, and quality checks were done on the Biomics sequencing platform of the Institut Pasteur (C2RT) (Paris, France) by short reads (paired-end, 250 bp length) on a MiSeq system (Illumina, San Diego, U.S.).

Bacterial contamination in the AMAZONIE culture was minimised by filtration, and DNA and RNA were extracted using standard protocols detailed in Additional file 2: Methods S4. Nanopore sequencing was performed using 4 μg of genomic DNA. The DNA was sheared at 20 kbp using Covaris g-TUBE (Covaris) according to the manufacturer’s protocol. After shearing, two libraries were prepared using a Ligation Sequencing Kit from Oxford Nanopore Technologies (SQK-LSK108). The prepared library was loaded onto a R9.4.1 Spot-On Flow cell (FLO-MIN106). Sequencing was performed on a MinION Mk1B machine for 48 h using the MinKNOW 2.0 software. Basecalling was performed using Guppy 3.0.3 with the Flip-flop algorithm. Illumina sequencing of the genomic DNA was performed using 1 μg of genomic DNA with Illumina HiSeq 2000 (2 × 150 bp) paired-end technology, with libraries prepared using TruSeq DNA PCR-Free (Illumina, San Diego, CA), at Macrogen Inc. (Seoul, South Korea). The transcriptome was sequenced using HiSeq 2000 (2 × 100 bp) paired-end technology, with libraries prepared using the TruSeq RNA sample prep kit v2 (Illumina, San Diego, CA), at Macrogen Inc. (Seoul, South Korea).

Genomic DNA from P. patens was fragmented to ~300 bp average length on a Covaris S220 (Covaris) with a micro TUBE AFA Fiber Snap-Cap (Covaris), and subsequently subjected to size selection by using SPRI beads (Beckman Coulter). Libraries were prepared from the fragmented DNA using TruSeq DNA PCR-free (Illumina). The libraries were sequenced with 150 bp paired-end reads on an Illumina HiSeq X (Illumina).

Libraries using 1100 ng gDNA for WGS were prepared with TruSeq DNA PCR-Free (Illumina, San Diego, CA, US) for all patients except for Pat5 and Pat9. Due to limited sample availability, the libraries for Pat5 and Pat9 were prepared with 200 ng input gDNA using NxSeq AmpFREE Low DNA Library Kit (Lucigen, Biosearch Technologies, UK). Briefly, gDNA was fragmented with Covaris E220 (Covaris, MA, US) to obtain insert size of approximately 350 base pairs (bp). The fragments were end-repaired and a-tailed, followed by ligation of unique dual index adaptors (Illumina). Library quantification was performed by KAPA Library Quantification Kit (Roche) and sequencing was done on a NovaSeq 6000 system (Illumina) using paired-end 150 bp readout, aiming at 300 million (M) paired reads.