Magattract hmw dna kit

Genomic DNA was extracted from the 142 blood samples using the QIAGEN MagAttract HMW DNA Kit (Qiagen Hilden, Germany). The concentration of the extracted DNA was measured using the qubit fluorometer (Life Technologies, United Kingdom) and concentrations greater than 25 ng/μL were accepted for further analysis. DNA integrity was verified on a 1% agarose gel. High-quality DNA was genotyped using the Illumina Bovine HD BeadChip Array kit (Illumina, Inc., San Diego, CA, United States) and in accordance with the Infinium HD Assay Super Manual Protocol at the Agricultural Research Council Biotechnology Platform, South Africa.
SNP quality control was conducted using PLINK version 1.9 (Chang et al., 2015 (link)). A genotyping rate for individual animals’ assessment was set at 0.95. SNPs were pruned based on a genotyping call rate of 0.95, Hardy-Weinberg < 0.00001, minor allele frequency <0.05. In addition, SNPs without chromosomal positions were removed from the data. The call rate of 0.95 removed 2 animals and 140 animals remained for downstream analysis. The assessment of SNP quality removed 8,463 SNPs for the genotyping call rate, 4,259 SNPs for Hardy-Weinberg and 142,290 SNPs for minor allele frequency. SNPs without chromosomal position (n = 847) were also removed and 622,103 SNPs remained for downstream analysis.

The same genome reference inbred domestic cat, Cinnamon, the Abyssinian, was used for the long-read sequencing [84 (link), 35 (link)]. High molecular weight DNA was isolated using a MagAttract HMW-DNA Kit (Qiagen, Germantown, MD) from cultured fibroblast cells according to the manufacturer’s protocol. Single molecule real-time (SMRT) sequencing was completed on the RSII and Sequel instruments (Pacific Biosciences, Menlo Park, CA).

Genomic DNA was extracted from ‐ 80°C frozen blood sample of a young male barn owl (M026801) using MagAttract HMW DNA Kit (Qiagen). In total five extractions quantified on a Qubit Fluorometer (ThermoFisher Scientific) yielded each between 1 and 6 µg of genomic DNA of a length between 35 and 50 kb (Fragment analyzer, Advanced Analytical, Labgene). In order to obtain a high‐quality de novo assembly, we combined libraries generated from standard short insert paired‐end libraries with that from mate‐pair libraries and PacBio long sequencing. Two mate‐pair libraries of 2 and 5 kb were prepared and sequenced by Fasteris (Fasteris), three TruSeq paired‐end libraries of 180 and twice 500 bp were prepared and sequenced at the Genomic Technologies Facility (GTF, University of Lausanne). The same high molecular weight DNA was used for PacBio libraries at the GTF. For PacBio, the DNA was sheared in a Covaris g‐TUBE (Covaris) to obtain 20 kb fragments. Then, the DNA size distribution was checked on the Fragment analyzer. 5 µg of the sheared DNA was used to prepare a SMRTbell library with the PacBio SMRTbell Template Prep Kit 1 (Pacific Biosciences) according to the manufacturer's recommendations. The library was sequenced on 37 SMRT cells with P4/C2 chemistry and MagBeads on a PacBio RSII system (Pacific Biosciences) at 240 min movie length.

High molecular weight genomic DNA of Nalm6 (ATCC, Manassas, VA, USA; the same origin below) was extracted using Qiagen MagAttract HMW DNA Kit. 1.0 ng HMW gDNA was input, and each molecule was captured by a gel bead which contained specific barcode to distinguish different molecules on 10X Genomics Chromium Controller, then amplified by isothermal incubation. The barcoded DNA was purified, then end repaired, dA-tailed and adaptor ligated using chromium genome library kit. Finally, the library was purified and enriched by index PCR amplification then sequenced paired-end 151 cycles on Illumina HiSeq4000.
The Long Ranger 2.0.0 pipeline (https://support.10xgenomics.com/genome-exome/software/pipelines/latest/using/wgs) was used to perform variant calling and phasing. First, we mapped the reads to hg19 with Lariat using default parameters. Second, Freebayes v0.9.21 was used to call haplotype-based variants. Finally, we filtered the variants using bcftools v1.1 (-e ‘(%QUAL<= 15 || (AF[0]>0.5 && %QUAL<50))’ and -e ‘(AO[0]<2 || AO[0]/(AO[0]+RO) <0.15)’).

We performed bulk Circle-Seq according to the previously described protocol^{7 (link), 10 (link)}, including the following modifications. In brief, we harvested 1 million cells during the exponential growth phase and extracted high molecular weight genomic DNA with the MagAttract HMW DNA Kit (Qiagen, cat. no. 67563). Next, we digested 1 µg of DNA in 100 µL of digestion mix containing 20 U of Plasmid-Safe ATP-Dependent DNase (Lucigen, cat. no. E3101K), 25 mM ATP (Lucigen, cat. no. E3101K), 1X Plasmid-Safe Reaction Buffer (Lucigen, cat. no. E3101K) for 6 days at 37 °C. Every 24 h we replenished the enzymes in the digestion mix by adding 2 µL of fresh Plasmid-Safe ATP-Dependent DNase, 4 µL of ATP, and 0.6 µL of Plasmid-Safe 10X Reaction Buffer. After 6 days, we purified circular DNA with 1X AMPure XP beads (Beckman Coulter, cat. no. A63881) following the manufacturer’s instructions. Lastly, we used 20 ng of purified circular DNA as input for the Nextera XT DNA Library Preparation Kit (Illumina, cat. no. FC-131-1024) and sequenced the libraries on a HiSeq X Ten (Illumina) machine, aiming at generating around 10 million reads per sample.

Genomic DNA was isolated and purified using a MagAttract HMW DNA Kit (Qiagen, Germany). The concentration and purity of extracted DNA were determined using a Qubit 2.0 fluorometer (Invitrogen, USA). Whole-genome sequencing of strain DMLL10 was performed using Single-Molecule Real-Time (SMRT) sequencing system (10 kbp) on a PacBio Sequel platform (Pacific Bioscience, USA) by CJ Bioscience, Inc. (Korea). A total of 136,666 reads (5355.39 × coverage) were generated. These reads were assembled into one contig using CLC Genomics Workbench ver. 7.5.1(CLC Bio, Denmark) with the HGAP4 algorithm in SMRT Link (version 10.1.0; Pacific Bioscience). Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (version 4.6) [22 (link)]. Open Reading Frames (ORFs) were predicted using Glimmer 3 [23 (link)], followed by annotation through a search against Clusters of Orthologous Groups (COG) database [23 (link)].

DNA was extracted using the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany) and sequenced at the Sequencing Core at the University of Illinois at Chicago according to their standard protocol for NextSeq500 (Illumina Inc., San Diego, CA, USA). Long-read sequencing was carried out using the SQK-RBK004 kit (Oxford Nanopore Technologies (ONT), Oxford, UK) on the MinION (ONT) with Guppy software (ONT) according to the manufacturer’s instructions. Illumina reads were quality screened using Fastp [7 (link)], assembled with long reads using Unicycler [8 (link)], and annotated using Prokka [9 (link)]. Strain type was predicted using pubMLST’s C. freundii scheme (https://pubmlst.org/cfreundii/, accessed on 1 August 2020). ORFs were searched against the CARD and NCBI databases using DIAMOND BLAST [10 (link)]. Whole-genome sequencing (WGS) using both short-read and long-read technologies was used as previously described [11 (link)].