Genomic DNA from collected blood samples was quantified with a Qubit fluorescence assay (Thermo Fisher Scientific, Waltham, MA, USA) and sheared with an S2 instrument (Covaris, Woburn, MA, USA). Library preparation was conducted using the TruSeq DNA PCR-Free HT Kit (Illumina, San Diego, CA, USA). Individual DNA libraries were measured using the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) qPCR and Qubit (Thermo Fisher Scientific). All flow cells were sequenced on a HiSeq 2500 sequencer (Illumina) using the SBS kit V4 chemistry (Illumina). FastQC was used for quality control, and the resulting reads were aligned to the hg19 reference genome with the BWA-MEM algorithm. The identification of SNPs and indels and genotyping were performed across all samples simultaneously using standard hard-filtering parameters or variant quality score recalibration according to the GATK Best Practices recommendations. WGS was performed using a minimum median coverage of 30×.
Truseq dna pcr free ht kit
Manufactured by Illumina
Sourced in United States, China
The TruSeq DNA PCR-Free HT Kit is a library preparation solution for next-generation sequencing. It enables the construction of DNA libraries without the need for PCR amplification, which can introduce bias. The kit provides a streamlined workflow for generating high-quality libraries from DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Sbs kit v4 chemistry, by Illumina (4 mentions)
Hiseq 2500 sequencer, by Illumina (4 mentions)
S2 instrument, by Covaris (4 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Qubit fluorescence assay, by Thermo Fisher Scientific (3 mentions)
Paired end cluster kit v4, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Miseq reagent kit v2, by Illumina (2 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Oncomine comprehensive assay v3, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Cbot instrument, by Illumina (1 mentions)
Miseq 2500, by Illumina (1 mentions)
Qubit 3, by Thermo Fisher Scientific (1 mentions)
Qiaquick 96 pcr purification kit, by Qiagen (1 mentions)
Miseq fgx platform, by Illumina (1 mentions)
11 protocols using truseq dna pcr free ht kit
1
Whole Genome Sequencing of Blood Samples
2
Comparative Sequencing of Biological Samples
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Except for the four samples (2800M, components B and C of the 2391c standard reference material®, and M2) detected by both platforms in concordant studies, all other samples were sequenced on the MGISEQ-2000RS platform.
For MGISEQ-2000RS sequencing, libraries were prepared using the MGIEasy Amplicon Library Preparation Kit (MGI) as described in a previous publication [31 (link)], and sequenced using an MGISEQ-2000RS High-throughput Sequencing Kit (MGI) with a read length set at 350 bases. For Miseq FGx sequencing, libraries were prepared using the Truseq® DNA PCR-Free HT Kit (Illumina), and quantified using the KAPA Library Quantification Kit (Roche, Basel, Switzerland) on a 7500 realtime PCR system. The MiSeq v2 Reagent Kit (300 cycles PE; Illumina) was used for sequencing.
For MGISEQ-2000RS sequencing, libraries were prepared using the MGIEasy Amplicon Library Preparation Kit (MGI) as described in a previous publication [31 (link)], and sequenced using an MGISEQ-2000RS High-throughput Sequencing Kit (MGI) with a read length set at 350 bases. For Miseq FGx sequencing, libraries were prepared using the Truseq® DNA PCR-Free HT Kit (Illumina), and quantified using the KAPA Library Quantification Kit (Roche, Basel, Switzerland) on a 7500 realtime PCR system. The MiSeq v2 Reagent Kit (300 cycles PE; Illumina) was used for sequencing.
3
Whole Genome Sequencing Protocol
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Whole blood was collected for genomic DNA extraction. Genomic DNA was quantified with a Qubit fluorescence assay (Thermo Fisher Scientific) and sheared with an S2 instrument (Covaris). Library preparation was carried out using the TruSeq DNA PCR-Free HT Kit (Illumina). Individual DNA libraries were measured by 2100 Bioanalyzer (Agilent) qPCR and Qubit (Thermo Fisher Scientific). Normalized DNA libraries were combined into five-sample pools per flow cell in all eight lanes and clustered on a cBot instrument (Illumina) with Paired-End Cluster Kit V4 (Illumina). All flow cells were sequenced on the HiSeq2500 sequencer (Illumina) using the SBS Kit V4 chemistry (Illumina). FastQC was used to check read quality, and the resulting reads were aligned to the hg19 reference genome with the BWA-MEM algorithm (10 (link)). Single nucleotide variants (SNVs) and indel identification and genotyping were performed across all samples simultaneously using standard hard filtering parameters or variant quality score recalibration according to GATK Best Practices recommendations. WGS was presented with a minimum, median coverage of 30X.
4
Whole Genome Sequencing of Blood Samples
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Genomic DNA from collected blood samples was quantified with a Qubit fluorescence assay (Thermo Fisher Scientific) and sheared with an S2 instrument (Covaris). Library preparation was carried out using the TruSeq DNA PCR-Free HT Kit (Illumina). Individual DNA libraries were measured with 2100 Bioanalyzer (Agilent) qPCR and Qubit (Thermo Fisher Scientific). All flow cells were sequenced on a HiSeq 2500 sequencer (Illumina) using SBS kit V4 chemistry (Illumina). FastQC was used to check read quality, and the resulting reads were aligned to the hg19 reference genome with the BWA-MEM algorithm [34 ]. The identification of SNPs and indels and genotyping were performed across all samples simultaneously using standard hard filtering parameters or variant quality score recalibration according to GATK Best Practices recommendations [35 (link)].WGS was performed with a minimum, median coverage of 30X.
5
Mitogenome Assembly and Annotation
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High-throughput sequencing libraries were constructed using the Illumina TruSeq DNA PCR-Free HT Kit and sequenced on an Illumina HiSeq 2500 with the 250 bp paired-end strategy. The quality of the raw sequencing reads was assessed using FastQC version 0.11.9, and the adapter sequences and low-quality reads were removed using Trimmomatic version 0.39. The filtered sequencing reads were then assembled into a complete mitogenome using NOVOPlasty 4.2. The tRNAs’ typical clover-leaf secondary structure and anticodon were identified using tRNAscan-SE 2.0. Finally, the codon usage of protein-coding genes (PCGs) and the nucleotide composition of the mitogenomes were determined using MEGA 5.0.
6
Comprehensive Genomic Profiling of Cancer
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Genomic DNA (gDNA) was extracted from whole blood for the generation of a genome sequencing library and BRCA1/2 targeted library using the TruSeq® DNA PCR-Free HT Kit (Illumina, Inc., San Diego, CA, USA) and OncomineTM BRCA Research Assay (Thermo Fisher Scientific, Waltham, MA, USA), respectively. gDNA was obtained from primary ovarian, metastatic peritoneum, and bilateral breast cancer tissues to detect somatic variants with the OncomineTM Comprehensive Assay v3 (Thermo Fisher Scientific). The resulting libraries were quantified and qualified with an InvitrogenTM QubitTM 3 Fluorometer (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer system (Agilent Technologies, Inc., Santa Clara, CA, USA), respectively. The libraries were then used for massively parallel sequencing analysis with various average sequencing depths of 30× for genome sequencing, 500× for the BRCA1/2 assay, and 1500× for the OncomineTM Comprehensive Assay v3.
7
SIV Env V1 Region Amplicon Sequencing
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Env V1 region amplicons from plasma and CSF SIV RNA and tissue SIV DNA were generated by PCR using nested primers as previously described [25 (link)]. Amplicons were tagged using the TruSeq DNA PCR free HT kit (Illumina, San Diego, California, USA). Successfully tagged products were purified with AmPure XP beads (Beckman Coulter, Brea, California, USA), quantified, and pooled. Approximately 14 pm of pooled DNA was then loaded onto a 600-cycle MiSeq Reagent Kit V3 on an Illumina MiSeq. FASTQ reads were then imported into Geneious V 7.1.7 (Biomatters, Ltd, Auckland, New Zealand). Reads were trimmed, paired, and merged using fast length adjustment of short reads (FLASH). Merged reads were mapped back to a consensus sequence generated from a stock of this virus. Reads spanning the amplicon of interest were extracted, and only those of sizes 450–500 bp were considered. Identical duplicates were detected and frequencies were calculated. For the phylogenetic analysis, reads were mapped to the reference sequence for the V1 portion of SIV/DeltaB670 env, using Geneious V 8.1.7. After de-novo assembling, contiguous reads were quantitated and extracted for analysis. Results are presented as nucleotide or amino acid sequence alignment assessed by ClustalW and phylogenetic UPGMA trees using Neighbor-joining consensus genetic distance model.
8
Whole Genome Sequencing of Human Samples
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Whole blood was collected for genomic DNA extraction. Genomic DNA was quantified with a Qubit fluorescence assay (Thermo Fisher Scientific) and sheared with a S2 instrument (Covaris). Library preparation was carried out using the TruSeq DNA PCR-Free HT kit (Illumina). Individual DNA libraries were measured by 2100 Bioanalyzer (Agilent) qPCR and Qubit (Thermo Fisher Scientific). Normalized DNA libraries were combined into five-sample pools per flow cell in all eight lines and clustered on a cBot instrument (Illumina) with Paired-End Cluster Kit V4 (Illumina). All flow cells were sequenced on the HiSeq. 2500 sequencer (Illumina) using SBS kit V4 chemistry (Illumina). FastQC was used to check read quality, and the resulting reads were aligned to the hg19 reference genome with the BWA-MEM algorithm16 . SNV and indel identification and genotyping were performed across all samples simultaneously using standard hard filtering parameters or variant quality score recalibration according to GATK Best Practices recommendations17 (link),18 . WGS was performed with a minimum median coverage of 30X.
9
Mitochondrial Genome Sequencing Pipeline
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The mitochondrial genome sequences were gained by next-generation sequencing. Prior to library construction, the DNA was quantified by Qubit 3.0 (Invitrogen, Life technologies, Carlsbad, CA, USA). The library with two indexes was constructed using the Illumina TruSeq@ DNA PCR-Free HT Kit and sequenced by BerryGenomics Company (Beijing, China) using Illumina Miseq 2500 with the strategy of 250 paired-ends.
The mitochondrial data constitutes a small fraction of huge primary data generated by genomic sequencing (approximately 0.5%) [11 (link),47 (link)]. To simplify the de novo assembly of mitochondrial genome from short reads produced, the mitochondrial targets were filtered at the stage of raw reads by similarity searches against a database of hymenoptera mitochondrial genomes, using BLASTn version 2.2.27+ with the E value of 1 × 10−5 and maximum target sequences of 1. Putative mitochondrial reads allowing for blast hits were extracted with a Perl script (FastqExtract.pl) [41 (link)]. All putative mitochondrial reads from the library were assembled into contigs with Celera Assembler version 8.3rc2 and IDBA version 1.1.1 as described in [15 (link)]. The de novo assembly of the mitochondrial contigs generated in previous methods were conducted by Geneious version 9.1.4 [70 (link)].
The mitochondrial data constitutes a small fraction of huge primary data generated by genomic sequencing (approximately 0.5%) [11 (link),47 (link)]. To simplify the de novo assembly of mitochondrial genome from short reads produced, the mitochondrial targets were filtered at the stage of raw reads by similarity searches against a database of hymenoptera mitochondrial genomes, using BLASTn version 2.2.27+ with the E value of 1 × 10−5 and maximum target sequences of 1. Putative mitochondrial reads allowing for blast hits were extracted with a Perl script (FastqExtract.pl) [41 (link)]. All putative mitochondrial reads from the library were assembled into contigs with Celera Assembler version 8.3rc2 and IDBA version 1.1.1 as described in [15 (link)]. The de novo assembly of the mitochondrial contigs generated in previous methods were conducted by Geneious version 9.1.4 [70 (link)].
10
Illumina MiSeq DNA Library Preparation
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The PCR products were purified with the QIAquick 96 PCR Purification Kit (Qiagen) and the TruSeq DNA PCR-Free HT Kit (Illumina, San Diego, CA, USA) and used for library preparation, according to the manufacturer’s guidelines. The libraries were sequenced on a MiSeq FGx platform (Illumina) using the Miseq Reagent Kit v2 (Illumina), with a read length of 250 bases.
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