Sequencing libraries were prepared from genomic DNA for Illumina MiSeq and HiSeq2500 platforms. A short insert (330 bp) paired-end (PE) library was constructed using a TruSeq DNA PCR-Free LT Sample Prep Kit (Illumina), which reduced PCR amplification bias in library preparation. Mate-paired (MP) libraries with various insert sizes (2, 4, 6, and 8 kbp) were constructed using the Nextera Mate Pair Sample Prep Kit (Illumina). The PE library was sequenced using MiSeq (2 × 230 bp) and the four MP libraries were sequenced using HiSeq2500 (2 × 100 bp).
Truseq dna pcr free lt sample prep kit
Manufactured by Illumina
Sourced in United States
The TruSeq DNA PCR-Free LT Sample Prep Kit is a library preparation kit designed for Illumina sequencing platforms. It enables the generation of sequencing libraries from DNA samples without the need for PCR amplification, providing a more direct and streamlined approach to library construction.
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Lab products found in correlation
Hiseq 2500, by Illumina (6 mentions)
Nextera mate pair sample prep kit, by Illumina (4 mentions)
Bluepippin, by Sage Science (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Miseq platform, by Illumina (2 mentions)
Genome analyzer iix platform, by Illumina (1 mentions)
Truseq dna sample prep kit, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Gentra puregene tissue kit, by Qiagen (1 mentions)
Large construct kit, by Qiagen (1 mentions)
Hiseq x hd pe cluster kit, by Illumina (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Magattract hmw dna kit, by Qiagen (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Genomic dna buffer set, by Qiagen (1 mentions)
100 g genomic tips, by Qiagen (1 mentions)
Kapa dna library quantification kit, by Roche (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
18 protocols using truseq dna pcr free lt sample prep kit
1
Illumina Sequencing Library Preparation
2
Whole Genome Sequencing of Caenorhabditis elegans Strains
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Worms for genome extraction were cultured in NGM under well-fed conditions at 20 °C. High quality genomic DNA was extracted using a Gentra Puregene Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The genome sequences of KR314, RC301, CB4854, and N2 were determined on an Illumina Genome Analyzer IIx platform using an Illumina TruSeq DNA Sample Prep Kit (Illumina, San Diego, CA, USA), which generated from 100 to 140 million reads; each read length was 110 bp. The genome sequences of AB1, CB4852, CB4853 were determined on an Illumina Hiseq 2500 platform using a TruSeq DNA PCR-Free LT Sample Prep Kit (Illumina), which generated from 50 to 130 million reads; each read length was 150 bp. Over 90 % of the genome was sequenced at a 10X coverage, except for CB4853 in which about 30 % was sequenced at 10X coverage. The nucleotide sequences reported in this paper have been submitted to the DDBJ Sequence Read Archive under accession numbers DRA002599 and DRA004249.
3
BAC Clone DNA Sequencing with Illumina
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Circular BAC clone DNA was prepared using a QIAGEN Large-Construct kit according to the supplier’s procedure, in which an exonuclease digestion procedure is included (QIAGEN). For nucleotide sequence determination of BAC clone DNAs, an Illumina Miseq was used. Libraries were prepared according to a protocol provided by the manufacturer, with slight modifications. Fragmented BAC clone DNA was further purified using Blue Pippin (Sage Science). A paired-end library consisting of clones containing ∼720 bp insert DNA fragment was prepared for the Miseq using a TruSeq DNA PCR-Free LT Sample Prep Kit (Illumina). Adapter sequences were removed from all sequence reads using Trimmomatic-0.30 [21 (link)]. Paired-end reads of high quality (quality-value ≥20) were assembled de novo using Newbler 2.9 (GS Assembler) to create a scaffold. From the scaffold, vector sequence was removed, and genomic sequence was extracted.
4
Genome Assembly Using Illumina Sequencing
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The Illumina platform (Miseq and Hiseq 2500) was used for sequencing.13 (link) Libraries were prepared according to slight modifications of protocols provided by the manufacturer. Fragmented genomic DNA was further purified using Blue Pippin (Sage Science). A paired-end library consisting of clones ∼720 bp was prepared for the Miseq using a TruSeq DNA PCR-Free LT Sample Prep Kit (Illumina), and 3-kb and 8-kb mate-pair libraries were prepared for the Hiseq 2500 using a Nextera Mate Pair Sample Prep Kit (Illumina), respectively (Supplementary Table S1 ). Longer reads were obtained by using more reagent kits for the Hiseq. K-mer counting and estimation of genome size were performed using JELLYFISH 2.2.0 software.14 (link),15 (link)
Adapter sequences were trimmed from all reads using Trimmomatic-0.30.16 (link) Paired-end reads of high quality (quality value ≥ 20) were assembled de novo using Newbler 2.9 (GS Assembler) to create contigs. Then subsequent scaffolding of the Newbler output was performed using SSPACE 3.0,17 (link) based on Illumina mate-pair information. Gaps inside scaffolds were closed using GapCloser 1.12.18 (link) Diploid sequences of gap-closed scaffolds were merged with Haplomerger-2-20151124.19 (link) CEGMA 2.5 software20 (link) was used to evaluate genome assembly. The mitochondrial genome was generated with the IDBA_UD 1.1.1 assembler.21 (link)
Adapter sequences were trimmed from all reads using Trimmomatic-0.30.16 (link) Paired-end reads of high quality (quality value ≥ 20) were assembled de novo using Newbler 2.9 (GS Assembler) to create contigs. Then subsequent scaffolding of the Newbler output was performed using SSPACE 3.0,17 (link) based on Illumina mate-pair information. Gaps inside scaffolds were closed using GapCloser 1.12.18 (link) Diploid sequences of gap-closed scaffolds were merged with Haplomerger-2-20151124.19 (link) CEGMA 2.5 software20 (link) was used to evaluate genome assembly. The mitochondrial genome was generated with the IDBA_UD 1.1.1 assembler.21 (link)
5
Illumina MiSeq Sequencing of PCR Products
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Library building was performed on the purified pools of PCR products using the TruSeq DNA PCR-free LT Sample Prep kit (Illumina). A total of four libraries (corresponding to the four pools) were constructed. Each library thus included one replicate of every sample, two PCR blanks, and one mock sample replicate. Additionally, each library contained one replicate of each of eight samples from another research project. The manufacturer’s protocol was followed with the exception that samples were incubated with the elution buffer over two rounds of 37°C for 10 minutes. Approximately 250 ng of PCR product from each pool was used as input for the libraries, and a library blank was included. The concentration and fragment size distribution of the libraries were verified on an Agilent 2100 Bioanalyzer. Libraries were pooled in equimolar concentrations and sequenced on the Illumina MiSeq platform (½flow cell) at the Danish National Sequencing Centre, applying 150 bp Paired-End sequencing. A spike-in of PhiX was used to increase complexity in the runs.
6
PCR-Free DNA Library Preparation
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A PCR-free library is preferable to minimize the base-composition bias in bulk samples and the already-existing PCR amplification bias in single-cell samples. Thus, PCR-free libraries were generated from 1.0 µg of genomic DNA from all bulk samples and 1.5 µg of amplified DNA from a single cell, based on their ladder-like distribution base sizes, using an Illumina TruSeq DNA PCR-Free LT Sample Prep kit (Illumina, San Diego, CA, USA; cat. no. FC-121-3001) according to the manufacturer's instructions. The DNA was sonicated into fragments with an average size of 350 bp, followed by end-repair, A-tailing and ligation with full-length adapter for Illumina sequencing but without further PCR amplification. The final libraries were evaluated using an Agilent 2100 Bioanalyzer and quantified by real-time PCR. Libraries were clustered on the cBot Cluster Generation System using a HiSeq X HD PE Cluster kit (Illumina) and subsequently sequenced on the Illumina HiSeq X Ten platform (paired ends, 150 bp).
7
Genome Assembly of Testis DNA via Illumina Sequencing
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Genomic DNA was extracted from the testis using a Mag attract HMW DNA kit (Qiagen, Santa Clarita, CA, USA). Paired-end (250 bp) and mate-pair libraries (3, 6, 10, and 15 kb) were constructed using a TruSeq DNA PCR-Free LT Sample Prep Kit and a Nextera Mate Pair Sample Prep Kit (Illumina, San Diego, CA, USA). Sequencing employed an Illumina HiSeq2500. We generated 731,873,830 bp of raw sequence data for de novo genome assembly. Genome assembly into scaffolds was performed using the Platanus v1.2.4 assembler54 after removal of adapter sequences (Platanus_trim and Platanus_internal_trim). Contig assembly was performed using only the PE library, and then scaffolding and gap closure were performed using all libraries. Genome Scope25 (link) was used for genome size and heterozygosity analyses. Generated scaffolds were named in order of length as pvir_s00001, starting with the longest.
8
NGS Library Preparation Protocol
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Library building was performed on the purified pools of PCR products using the TruSeq DNA PCR‐free LT Sample Prep kit (Illumina). A total of eight libraries (corresponding to the four PCR replicates from each sample for each of the two primer sets) were constructed (Supporting Information Figure S1 ). Each pool thus included one replicate of every sample, four DNA extraction blanks, two PCR blanks, and two mock sample replicates.
The manufacturer's protocol was followed with the exception that samples were incubated with the elution buffer over two rounds of 37°C for 10 min. Approximately 750 ng of PCR product from each pool was used as input for the libraries, and a library blank was included. The concentration and fragment size distribution of the libraries were verified on an Agilent 2100 Bioanalyzer. Libraries were pooled in equimolar concentrations and sequenced on one mid‐output flow cell on an Illumina NextSeq 500 (150 bp paired‐end sequencing) at the Biotech Research and Innovation Centre (BRIC), Dept. of Biology, University of Copenhagen. A spike‐in of 10% PhiX was used to increase complexity in the runs.
The manufacturer's protocol was followed with the exception that samples were incubated with the elution buffer over two rounds of 37°C for 10 min. Approximately 750 ng of PCR product from each pool was used as input for the libraries, and a library blank was included. The concentration and fragment size distribution of the libraries were verified on an Agilent 2100 Bioanalyzer. Libraries were pooled in equimolar concentrations and sequenced on one mid‐output flow cell on an Illumina NextSeq 500 (150 bp paired‐end sequencing) at the Biotech Research and Innovation Centre (BRIC), Dept. of Biology, University of Copenhagen. A spike‐in of 10% PhiX was used to increase complexity in the runs.
9
Whole Genome Sequencing DNA Shearing
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For all lines save one, two micrograms of genomic DNA were sheared to ∼550 bp using a Covaris E220 with settings: duty cycle 10%; intensity 175; cycles/burst 200; and time 80s. Only one microgram of DNA was available for line SIP_L2_2, so the DNA was sheared to ∼350 bp using a Covaris E220 with settings: duty cycle 10%; intensity 3; cycles/burst 200; and time 60s. Libraries were constructed using the Tru-Seq DNA PCR-Free LT Sample Prep Kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. The libraries were pooled and run on an Illumina HiSeq 2500 with version 3 sequencing reagents to generate a minimum of 10 million paired-end 251-base reads per library (Illumina, San Diego, CA), resulting in 30-50X genome coverage on average (Figure S1). The HiSeq data were processed using RTA1.18.64 and CASAVA 1.8.2.
10
Whole-Genome Sequencing of Cyanobacterial Strains
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Total DNA of PCC6803 and tolerant strains were extracted using the Genomic DNA Buffer Set and QIAGEN Genomic-tips 100/G (Qiagen, CA, USA). A DNA library was prepared using Truseq DNA PCR-Free LT sample prep kit (Illumina, CA, USA) and KAPA DNA library quantification kit (KK4824, KAPA Biosystems, MA, USA), and the sample was sequenced using MiSeq sequencer with MiSeq Reagent kit v2 (Illumina) generating 150 bp paired-end reads. The sequence data were mapped to the genome sequence of PCC6803 GT-I strain (NCBI Reference Sequence No.: NC_017038.1)27 (link) using Bowtie 2 software ver. 2.2.3 with default parameters41 (link). SNPs were identified using SAMtools ver 1.0 and BCFtools ver. 1.042 (link) with a threshold of a quality score of >150. The sequence data obtained from MiSeq was deposited in DDBJ Sequence Read Archive under accession number DRA010198. The mutations identified by whole-genome sequencing were confirmed by Sanger sequencing using DNA fragments, including the mutation site amplified by PCR using the primer pair listed in Supplementary Table 4 .
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