To isolate genomic DNA, each individual adult zebrafish was euthanized and placed in proteinase K digestion buffer overnight, followed by phenol chloroform extraction and ethanol precipitation, using previously described methods50 . Prior to genomic sequencing, carryover organics were removed from the genomic DNA using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s instructions. We used a single-library per sample approach for high-throughput sequencing. Briefly, Illumina TruSeq DNA PCR-free libraries were constructed from genomic DNA isolated from each individual zebrafish. To facilitate Discovar de novo assemblies, the libraries were individually sequenced in single lanes on a HiSeq2500 instrument (Rapid run mode), using paired-end 2 × 250 bp reads, providing approximately 50–60 × coverage.
Truseq dna pcr
Manufactured by Illumina
Sourced in United States
The TruSeq DNA PCR is a library preparation kit designed for whole-genome sequencing. It enables the construction of DNA libraries from genomic DNA samples. The kit includes reagents and protocols for fragmentation, end-repair, A-tailing, and adapter ligation. The kit is optimized for use with Illumina sequencing platforms.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (4 mentions)
Clc genomic workbench 11, by Qiagen (2 mentions)
Truseq nano dna library prep, by Illumina (2 mentions)
Ffpe extracted genomic dna sample preparation, by Illumina (2 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Hiseq reads, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Truseq sbs kit v3, by Illumina (1 mentions)
Novaseq 6000, by Macrogen (1 mentions)
Hiseq x sequencer, by Illumina (1 mentions)
Fastdna spin kit for soil, by MP Biomedicals (1 mentions)
Hiseq x10 platform, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Qiagen (1 mentions)
Rna easy mini kit columns, by Qiagen (1 mentions)
Trim galore v0, by Babraham Bioinformatics (1 mentions)
Fastqc v0, by Babraham Bioinformatics (1 mentions)
Hiseq x platform, by Illumina (1 mentions)
Powersoil kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
18 protocols using truseq dna pcr
1
Zebrafish Genomic DNA Isolation
2
Assembly and Analysis of Highly Repetitive Genomic Regions
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A phrap assembly (http://www.phrap.org/phredphrapconsed.html ; phrap version 1.090518) was generated from a randomly selected subset of Illumina HiSeq reads (40,000 paired-end 250 base reads), from an Illumina TruSeq DNA PCR-free library of H. echinata gDNA using the following command: phrap -ace test20kreadpairs -retain_duplicates -minscore 140 -minmatch 70 -vector_bound 0 -repeat_stringency .999 -forcelevel 0. The largest contig, out of a total of 11,790 contigs, included a complete representation of one example copy of the histone region (5998 bases). The second and third largest contigs were joined together in consed [36 (link)] using overlap information forming a complete representation of one example copy of the ribosomal DNA (rDNA) repeat region (7039 bases). Using a 17-base-long k-mer word use histogram from 31.1 × 106 paired-end 250 base reads, k-mers from the histone region appear at approximately 28,000-fold coverage and the rDNA repeat region appears at the approximately 46,000-fold coverage (Additional file 1 : S1B). With the diploid peak at 20× coverage (Additional file 1 : S1A), this indicates there are 1400 copies of the histone region and 2300 copies of the rDNA repeat region in a diploid nucleus.
3
Whole Genome DNA Sequencing of Bull Breeds
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Whole genome DNA sequences were generated as described in [40 (link)]. In brief: DNA was isolated from blood samples of 155 bulls using a DNA Isolation System, then libraries were generated from 1 μg of genomic DNA using the Illumina TruseqDNA PCR, and sequenced on the IlluminaHiSeq2000 with a 100 cycles of paired-end sequencing module using the Truseq SBS kit v3. All animals were selected and sequenced within the frame of the Gene2Farm project and represented 13 breeds: Brown Swiss (48), Fleckvieh (31), Norwegian Red (26), Guernsey (20), Simmental (16), Parda de la Montaña (4), Pezzata Rossa Italiana (3), Avileña (2), Bruna Italiana (1), Albera (1), Rubia Gallega (1), Toro de Lidia (1) and Pirenaica (1). The total number of raw reads obtained for a single bull varied between 83,423,880 (a Norwegian Red bull) and 763,594,929 (a Brown Swiss bull). The number of reads per individual was shown on Additional file 3 : Figure S3 The length of single read was 101 bp and the corresponding insert size was 350 bp. Data were paired-end type and the average quality of reads per bull ranged from 28.11 to 36.69.
4
Whole Genome Sequencing of APKS Organoids
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At passage 4 and passage 21 WGS was performed on the APKS organoids. At passage 4 a mix of DNA from the three replicates was used, while at passage 21 each replicate was sequenced individually. For this DNA was isolated from cells that were left over after passaging the culture. Library preparation and whole genome sequencing was performed at Macrogen using Illumina TruSeq DNA PCR free library preparation and sequenced on a HiSeq 10X with 2 × 150 bp paired end sequencing. Reads were aligned to GRCh38 using Burrows Wheeler Aligner v0.7.14 mapping tool with settings ‘bwa mem –M’35 (link). Duplicate reads were marked using Sambamba (version 0.6.6) dedup. Base Quality Score Recalibration was done using GATKBaseRecalibrator v3.7.36 (link) Somatic variants were detected using Mutect 2.2.37 (link)
5
Assembly of C. orientalis Draft Genome
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The draft genome from the C. orientalis accession 2007–03 (Figure 1—source data 1 ) was assembled from long reads generated by PacBio single-molecule real-time sequencing. Long reads were assembled with Falcon (Chin et al., 2016 (link)) (version 0.5.4, max_diff = 150, max_cov = 150, min_cov = 2). The resulting primary contig set was iteratively polished with Quiver again using long reads (Chin et al., 2013 (link)) (version 2.0.0) and with Pilon (Walker et al., 2014 (link)) (version 1.16) using short reads from a single Illumina TruSeq DNA PCR-free library. The draft genome of C. orientalis comprises 135 Mb distributed over 423 gap-free contigs and covers 60% of the C. rubella reference with non-ambiguous 1-to-1 whole genome alignments. Its completeness is comparable to that of the C. rubella reference.
6
Whole-Genome Sequencing of Yersinia pseudotuberculosis
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Purified total DNA was sequenced using the Illumina TruSeq DNA PCR-free protocol by Novogene. The sequencing reads were aligned to Y. pseudotuberculosis reference sequences (NC_010465 for the YPIII chromosome and an in-house pIB1 sequence for the plasmid) using the CLC Genomic Workbench 11 (CLC Bio; Qiagen). The coverage for single nucleotides was calculated and the average coverage in a 10-kb window was plotted for visualizations. The PCN and PTR were calculated by dividing the average coverage of plasmid DNA with the average coverage of chromosomal DNA and the average coverage at oriC with the average coverage at ter of the mapped reads, respectively. The sequence data used in this study have been deposited in the European Nucleotide Archive (ENA) under accession number PRJEB38239 .
7
Amplify and Sequence Bound Polynucleotides
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In order to amplify and sequence the bound polynucleotides, A RAVE based assay was performed. A poly (A) tail was introduced at the 3′end of the polynucleotides using TdT tailing reaction. 20 μl solutions containing dATP, 10 mM Tris–HCl, 50 mM KCl and 1.5 mM MgCl2 buffer, the extracted bound polynucleotide sequences (10 μl) and TdT 1–2 U/μl were prepared and incubated for 0.5–2 h followed by termination of the reaction by heating the solution to 75°C for 10 min. The resultant product was used directly as the template in PCR. 20 μl solutions containing 1× master mix (10 μl), 0.1–0.5 μM (1 μl) of the forward and reverse primers and 1–5 μl of the DNA sequences with poly (A) template were prepared. PCR reactions were performed over 30–40 cycles consisting of a denaturing step at 94°C for 30 s, an annealing step at 45–65°C for 30 s and an extension step of 72°C for 30 s. The resultant dsDNA libraries were sequenced using a NovaSeq 6000 (Macrogen, South Korea). All sample libraries were prepared using an Illumina TruSeq DNA PCR free library construction (Insert 350 bp) prior to sequencing with a pairwise read.
8
Whole Genome Sequencing of Pancreatic Cell Lines
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For whole genome sequencing, libraries of Panc0203, SP0926 and SP1030 genomic DNAs were prepared according to the Illumina TruSeq DNA PCR-free library preparation guide and sequenced using Illumina HiSeq X sequencer following quality control and quantification with 151nt read length, which was performed by Macrogen. After generation of raw data through an integrated primary analysis software called RTA 2 (Real Time Analysis 2), next-generation sequencing data was aligned by Isaac Aligner 01.15.02.08., the mapping reference was the hg19 from UCSC, and analyzed by IVC (Isaac Variant Caller 2.0.13), SnpEff 3.3, Control-FREEC 6.4, Manta 0.20.2 and Breakdancer.
9
TruSeq DNA PCR-Free library preparation
10
TruSeq DNA PCR-Free library preparation
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TruSeq DNA PCR-Free libraries were prepared from blood and FF tissues using 1 μg of input DNA according to the manufacturer’s instructions (Illumina, San Diego, CA). FFPE specimens were prepared using the Illumina FFPE-extracted genomic DNA sample preparation and TruSeq Nano DNA Library Prep (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions (Supplementary Methods ). Sequencing was performed on a HiSeq2500 (Illumina, San Diego, CA, USA) to an average depth of coverage of 70 × for tumor samples and 30 × for GL samples. Alignment is detailed in the Supplementary Methods .
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