To dissect the novel GWAS signal associated with AAO identified in the Spanish population, we performed whole-genome sequencing (WGS) analyses in 5 of 37 homozygous carriers of the PARK2 signal. DNA concentration was determined by Qubit fluorescence and normalized to 20 ng/uL. One microgram of total genomic DNA was sheared to a target size of 450 base pairs (bp) using the Covaris LE220 ultrasonicator. Library preparation was achieved using the TruSeq DNA PCR-Free High Throughput Library Prep Kit and IDT for Illumina TruSeq DNA UD Indexes (96 Indexes, 96 Samples). Sequencing libraries were assessed for size distribution, absence of free adapters, and adapter dimers on a Fragment Analyzer. Library quantitation was performed by quantitative polymerase chain reaction using the KAPA Library Quantification Kit subsequent normalization to 4 nM. Libraries were clustered on v2.5 flowcell using the Illumina cBot 2 System before sequencing on the Illumina HiSeq X System using paired-end 150-bp reads. BCL files processed with alignment by ISAAC on HAS 2.2 and BAMs were used for QC assessment of mean coverage, percent duplicates, percent bases >20× coverage, and percent noise sites.
Truseq dna pcr free high throughput library prep kit
Manufactured by Illumina
Sourced in United States
The TruSeq DNA PCR-Free High Throughput Library Prep Kit is a laboratory equipment product designed for the preparation of DNA libraries. It is a PCR-free approach to library construction, eliminating potential biases introduced by PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 4, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Kapa library quantification kit, by Illumina (1 mentions)
Le220 ultrasonicator, by Covaris (1 mentions)
Hiseq x system, by Illumina (1 mentions)
Cbot 2 system, by Illumina (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
Microtube, by Covaris (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
Nextseq, by Illumina (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mid output kit, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Phusion high fidelity pcr master mix with gc buffer, by New England Biolabs (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Bcl2fastq v2, by Illumina (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
13 protocols using truseq dna pcr free high throughput library prep kit
1
Whole-Genome Sequencing of PARK2 Carriers
2
P. heterophylla Leaf DNA Extraction and Sequencing
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The total genomic DNA from P. heterophylla leaf tissues was extracted using a modified CTAB method. DNA quantity and quality were determined using Qubit4.0 (Thermo Fisher Scientific Inc., USA). Subsequently, the genomic DNA was purified and fragmented to construct sequencing libraries (350 bp) using the TruSeq DNA PCR-Free High Throughput Library Prep Kit (Illumina, San Diego, CA). High-throughput sequencing (2 × 150 bp) was performed with the NovaSeq 6000 sequencer (Illumina, San Diego, CA).
3
High-throughput Bacterial DNA Extraction
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Frozen characterized strains were struck onto CHROMagar O157 (CHROMagar Microbiology) and grown overnight in 3.5 mL of tryptic soy broth (Becton Dickinson) at 37 °C for DNA isolation. One milliliter of growth media was used in the QIAamp DNA mini kit extraction kit (QIAGEN) according to manufacturer instructions with the addition of 4ul of RNase A (100 mg/ml) after lysis to each sample. DNA extractions were quantified and checked for purity using a Nanodrop 2000 spectrophotometers (Thermo Scientific) with 20 mL/ng as the minimum acceptable concentration. DNA (1.5 μg) from each sample was sheared to 350 bp with a Covaris Sonicator S220 (Covaris) using Covaris microtube (Covaris) and used as an input in the TruSeq DNA PCR-Free High Throughput Library Prep Kit (Illumina). DNA libraries were quantified via qPCR with the KAPA Library Quant Kit for Illumina libraries (Roche) and diluted to 4 nM prior to pooling (two pools of 96). Each pool was run on an Illumina NextSeq with the Mid-Output Kit (2 × 150).
4
Time-series Genomic Analysis of Evolving E. coli
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Genomic DNA from the starting culture and 24 samples of evolving bacterial populations in time series (6 reactors ×4 days) was extracted from cell pellets with the Sigma Aldrich GenElute Bacterial Genomic DNA kit (NA2110). Sequencing libraries were prepared with the Illumina TruSeq DNA PCR-Free High Throughput Library Prep kit (#20015963) according to the manufacturer’s instructions. DNA was sequenced with the Illumina NextSeq 500 using 2×150 reads to reach up to 500–1000-fold genomic coverage in each sample (see Table S1, available in the online version of this article, for data quality and statistics). Reads were cleaned from adapter sequences, poly-G sequences and were trimmed by Phred base quality score with the Trimmomatic [30 (link)] (with ‘SLIDINGWINDOW : 4 : 15 HEADCROP : 10 CROP : 145 MINLEN : 65′ parameters). Cleaned reads were mapped onto an E. coli
5
Whole Genome Sequencing of Mouse B Cells
6
Amplification and Sequencing of 16S rRNA Genes
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The Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, USA) was used for 16S rRNA amplification. 16S rRNA genes were amplified using the specific barcoded primers of 806R for V4 hypervariable regions. The PCR products were detected by electrophoresis using 2% agarose gel. The target band was extracted or cleaned using the QIAquick Gel Extraction Kit (QIAGEN, Germany). Finally, the TruSeq® DNA PCR-Free High-Throughput Library Prep Kit (Illumina, USA) was used for library construction, and quantification of a library was done using Qubit and real-time PCR. After the library was qualified, all samples were sequenced on the Illumina NovaSeq 6000 platform.
7
Plastid Genome Sequencing of Arnica Species
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Total genomic DNA extraction was performed on the leaf tissues using a modified CTAB method. The quantity and quality of the DNA were determined using Qubit 4.0 (Thermo Fisher Scientific Inc., USA). The sequencing library (~350 bp) was constructed using purified DNA and a TruSeq DNA PCR-Free High Throughput Library Prep Kit (Illumina USA). An Illumina NovaSeq platform was employed to conduct high-throughput sequencing. The raw data were deposited in the Sequence Read Archive (SRA) under BioProject accession number PRJNA682118. The final plastid genomes and concatenated nrDNA sequences of the A. lancea, A. chinensis, A. macrocephala, A. japonica, and A. koreana specimens were assembled, annotated, and submitted to GenBank (Supplementary Table 1
8
Whole-Genome Sequencing of Tumor and Blood Samples
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Following DNA extraction from tumor and matched peripheral blood samples, we constructed DNA libraries using a TruSeq DNA PCR-Free High Throughput Library Prep Kit (20015963; Illumina) with 1 μg DNA according to the manufacturer's instructions. Then we performed whole-genome 150-bp paired-end WGS using a NovaSeq 6000 System (Illumina). We converted the resultant raw data to FASTQ format with Bcl2fastq v2.20 (Illumina) and then used DRAGEN Bio-IT Platform v3.9 (Illumina) for mapping sequenced reads to reference human genome hs37d5, marking duplicated reads, variant calling, and calculating quality metrics.
9
High-throughput Whole-genome Sequencing Protocol
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Genomic DNA was extracted using NucleoSpin Plant II (Macherey-Nagel, Düren, Germany). After the genomic DNA was fragmented into approximately 350 bp fragments using a sonicator (E220 or LE220, Covaris, Woburn, MA, USA), sequencing libraries were synthesized using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA) or a TruSeq DNA PCR-Free High Throughput Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocols. Each DNA library was tagged with distinctive barcode sequences and equimolarly pooled into a tube. Of the 70 samples, 30 were sequenced in four lanes in HiSeq2500 (Illumina) with the paired-end 125 bp chemistry, while the remaining were sequenced in four lanes in HiSeq X Ten (Illumina) with the paired-end 150 bp chemistry. Raw sequence data are deposited in the DNA Data Bank of Japan (accession: DRA011715). We obtained an average of 10.3 G bases (range: 2.95–34.4 Gb) per sample.
10
Bacterial DNA Sequencing with Illumina MiSeq
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The DNA libraries from the mock community of 20 bacterial DNA sequences were prepared with the TruSeq® DNA PCR-Free High-Throughput Library Prep Kit (Illumina, San Diego, USA) following the protocol for the purified amplicons. Each 16S rRNA gene region amplification product was labeled with a different index. Sequencing was conducted in the Illumina MiSeq System using the MiSeq Reagent Kit v2 (Illumina, San Diego, USA) (Table S2).
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