Genomic DNA was extracted and 1 μg genomic DNA was treated with EZ DNA Methylation-Gold Kit (Zymo Research, D5005) according to the manufacturer’s instructions. The methylation-seq library was constructed using TruSeq Nano DNA High Throughput Library Prep Kit (Illumina, 20015965). The bisulfite-treated DNA methylation sequencing data were analyzed by a data analysis pipeline called Methy-Pipe44 . The paired-end reads were first aligned to the reference mouse genome (mm9) by the BSAligner module. The sequencing adaptors and low-quality bases on read ends were trimmed. All Cs in both the reference genome and sequenced reads were replaced by Ts in silico and the pre-processed and converted reads were aligned to the converted reference genome. Then methylation density (MD) calculation and the DMRs identification were performed by BSAnalyzer module. After MD calculation, a sliding window approach is employed and Mann–Whitney U test is recruited to identify differentially methylated seed regions (p-value <0.01). Regions with at least 20% changes of absolute methylation level and p < 0.01 were defined as DMRs. Finally, we defined the merged seed regions with significant difference as putative DMRs.
Truseq nano dna high throughput library prep kit
Manufactured by Illumina
Sourced in United States
The TruSeq Nano DNA High Throughput Library Prep Kit is a DNA library preparation solution designed for high-throughput sequencing on Illumina platforms. The kit provides a streamlined workflow for generating libraries from fragmented DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna mini kit, by Qiagen (2 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
Stool dna kit, by Omega Bio-Tek (1 mentions)
Miseq platform, by Illumina (1 mentions)
Novaseq 6 000 platforms, by Illumina (1 mentions)
Sequel sequencing kit 3, by Pacific Biosciences (1 mentions)
Pacbio smrtbell express template prep kit 2, by Pacific Biosciences (1 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
Pacbio sequel, by Pacific Biosciences (1 mentions)
Flexigene dna kit, by Qiagen (1 mentions)
Truseq stranded mrna sample prep kit, by Illumina (1 mentions)
7 protocols using truseq nano dna high throughput library prep kit
1
Mouse Genome Methylation Profiling
2
Evolutionary Analysis of Meropenem-Resistant S. pneumoniae
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To investigate evolutionary relationships of S. pneumoniae, we performed WGS on 27 isolates, including 24 meropenem-nonsusceptible isolates of serotype 15B/C-ST83 collected during 2013–2017 and 3 clinical isolates of serotype 23F-ST81 identified in our previous study (10 (link)). We used a QIAamp DNA Mini Kit (QIAGEN) to extract S. pneumoniae genomic DNA and prepared libraries for WGS sequencing by using the TruSeq Nano DNA High Throughput Library Prep Kit (Illumina, https://www.illimina.com ). We multiplexed library samples and sequenced on an Illumina MiSeq with 2×300-bp paired-end reads. We used SPAdes version 3.13.0 (19 (link)) to quality trim raw reads before assembling by using k-mer values 21–77 in careful mode. We used QUAST (20 (link)) to evaluate this assembly, which produced an average of 140 contigs with an N50 length in 101,632bp. Because S. pneumoniae PMEN1 ATCC-700669 (GenBank accession no. FM211187), a serotype of the 23F-ST81 strain, is genetically related to ST83, a single locus variant of ST81, we chose this strain as a reference sequence for mapping. We deposited WGS data, ≈1.4 million pair-end reads per sample, to the Sequence Read Archive database (SRA; http://www.ncbi.nlm.nih.gov/sra ) under accession nos. SRR8867342–68 (Appendix 1 Table 2).
3
Metagenomic Fecal DNA Extraction and Sequencing
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Total genomic DNA was extracted from the fecal samples using Stool DNA Kit (Omega Bio‐tek) according to the manufacturer's instructions. The fecal samples were inactivated at 56°C for 30 min before DNA extraction to inactivate SARS‐CoV‐2 viruses. The quantity and quality of the extracted DNA were assessed by Fluorometer (Promega) and agarose gel electrophoresis. For the qualified samples, the shotgun metagenomic libraries were constructed by the TruSeq Nano DNA High Throughput Library Prep Kit (Illumina). Read lengths were 2 × 150 bp with an insert size of ~400 bp. Dual indexed barcodes were applied for sample multiplexing. The prepared libraries were stored in a freezer at −20°C and sequenced by the Illumina HiSeq X Ten platform (Illumina).
4
Sequencing and Assembling H. pylori Genomes
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A genomic DNA sequencing of 135 new H. pylori strains was performed using the Miseq platform (Illumina, Inc., San Diego, CA, USA), allowing a paired-end (2 × 300 bp) sequencing strategy, and library preparation was done with the TruSeq Nano DNA High Throughput Library Prep Kit (Illumina, Inc., San Diego, CA, USA). After quality checking the raw reads using FastQC version 0.11.9 (https://github.com/s-andrews/FastQC , accessed on 23 August 2022), Trimmomatic pipeline version 0.39 was used to remove low-quality reads, resulting in averages of Q20. Then, we performed de novo assembly using Spades assembler implanted with Shovill version 1.1.0 (https://github.com/tseemann/shovill , accessed on 23 August 2022) and did quality assurance on all 223 draft genomes using the Quality Assessment Tool for Genome Assemblies (QUAST) version 5.2.0 [26 (link)]. All draft genomes were annotated for contigs > 500 bp with Prokka version 1.14.659 to identify features of interest in genomic DNA sequences [27 (link)].
5
Whole-Genome Shotgun Sequencing Workflow
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The DNA library was constructed using the Illumina TruSeq Nano DNA High Throughput Library Prep Kit (Illumina, USA) according to the manufacturer’s instructions. Whole-genome shotgun sequencing was performed using Novaseq 6000 platforms (Illumina, USA) with 2 × 150 paired-end reads. The raw reads were trimmed by removing adaptor sequences and low-quality sequences with Q < 20 using Trimmomatic v.0.39 with parameters of “SLIDINGWINDOW:4:20”72 (link), and MultiQC v.1.273 (link) summarized the sequence of quality control. The de novo genome assembly was carried out with SPAdes v.3.10.174 , and the assembly was polished with Pilon v1.2375 (link). The assembly was evaluated using QUAST v.4.576 (link), and the gVolante was used to assess the completeness of the genome assembly, according to the Eurotiomycetes database77 .
6
Whole Genome Sequencing using PacBio and Illumina
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Genomic DNA was extracted by grinding cells frozen in liquid nitrogen, followed by a conventional zymolyase-SDS treatment protocol from an overnight cultured sample in a yeast extract-peptone-dextrose (YPD) medium [25] (link). The overall quality of genomic DNA was checked using Tapestation 4200 (Agilent Technologies, Santa Clara, CA, USA) with gDNA Screentape (Agilent Technologies, Santa Clara, CA, USA). PacBio Sequel sequencing library was constructed using the PacBio SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA, USA) according to the manufacturer's instructions. Sequencing was performed using Sequel Sequencing Kit 3.0 and SMRT cells 1 M v3 Tray (Pacific Biosciences, CA, USA), and 600 min movies were captured for each SMRT cell using the PacBio Sequel (Pacific Biosciences, CA, USA) sequencing platform.
For Illumina Novaseq sequencing, libraries were prepared using TruSeq Nano DNA High Throughput Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Sequencing was performed using Illumina Novaseq (Illumina, San Diego, CA, USA) sequencing platform.
For Illumina Novaseq sequencing, libraries were prepared using TruSeq Nano DNA High Throughput Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Sequencing was performed using Illumina Novaseq (Illumina, San Diego, CA, USA) sequencing platform.
7
Comprehensive Genomic Profiling of Biliary Tract Cancers
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To evaluate HRD status, WGS was performed for BTCs (Fig. 1). First, we obtained WGS data from 36 All 36 BTCs were included in the germline variant analysis cohort. In addition, we selected 16 BTCs with HR-related pathogenic germline variants or VUS identified in the Hokkaido cohort. DNA was extracted from frozen tissue using the QIAamp DNA mini kit (QIAGEN) and the FlexiGene DNA Kit (QIAGEN) for blood, following the manufacturer's protocol. WGS libraries were prepared using the TruSeq Nano DNA High Throughput Library Prep Kit (Illumina) with 200 ng DNA input. 500-600 base pair (bp) insert libraries were prepared following the manufacturer's protocol. Thirty-five WGS libraries were sequenced on HiSeq 2500/2000, and 17 WGS libraries were sequenced on NovaSeq 6000, yielding paired reads of 100-150 bp. The mean sequence depths were 52x for the tumor WGS and 36x for the normal tissue WGS. We called germline and somatic variants from WGS data by using the integrated pipeline with HRD. 26 RNA was extracted from frozen tumor tissues, and RNA-seq libraries were prepared using the TruSeq Stranded mRNA Sample Prep kit (Illumina) following the manufacturer's protocol. We described further details regarding methods of WGS and RNA-seq analysis in the supplementary materials and methods.
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