The library was constructed using an Illumina TruSeq Nano DNA Low Throughput Library Prep Kit following the manufacturer’s instructions. Reads were mapped to the hg38 genome using bwa-0.7.15 (9 (link)). Indel realignment and base recalibration were performed using GATK-3.8 to improve indel detection sensitivity and correct base quality score bias (10 (link)). MuTect2 was used for single nucleotide variation (SNV) and insert-deletion (INDEL) calling. All variants were annotated using snpEff-2.3.7 (11 (link)). Tumor sample CNVs were called using Control-FreeC (version 9.5, parameters: ploidy = 2, breakPointThreshold = 0.5, coefficientOfVariation = 0.05, mateOrientation = FR) with paired blood samples as controls (12 (link)). GISTIC2 (version 2.0.23) was used to identify CNV regions that were significantly amplified or deleted across the ES or LS samples (parameters: -genegistic 1 -smallmem 1 -broad 1 -brlen 0.98 -conf 0.90 -savegene 1 -qvt 0.1) (13 (link)).
Truseq nano dna low throughput library prep kit
Manufactured by Illumina
Sourced in United States
The TruSeq Nano DNA Low Throughput Library Prep Kit is a library preparation solution designed for low-throughput sequencing. It is a core function of the kit to convert DNA samples into sequencing-ready libraries.
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Lab products found in correlation
Truseq dna single indexes set a, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Truseq dna single index set a and b, by Illumina (2 mentions)
Miseq reagent kit v2, by Illumina (2 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (2 mentions)
Miseq instrument, by Illumina (2 mentions)
Afa microtube, by Covaris (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Sample purification beads, by Illumina (1 mentions)
Qubit dsdna hs assay, by Thermo Fisher Scientific (1 mentions)
Hiseq x ten sequencing system, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Nucleospin microbial dna mini kit, by Macherey-Nagel (1 mentions)
Costar spin x, by Corning (1 mentions)
Sybr gold staining, by Thermo Fisher Scientific (1 mentions)
Platinum superfi pcr master mix, by Illumina (1 mentions)
Miniseq, by Illumina (1 mentions)
Nebnext dsdna fragmentase, by New England Biolabs (1 mentions)
Truseq dna pcr free low throughput library prep kit, by Illumina (1 mentions)
Qubit 3, by Thermo Fisher Scientific (1 mentions)
10 protocols using truseq nano dna low throughput library prep kit
1
Comprehensive Genomic Profiling Protocol
2
Diatom Genome Library Preparation
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Libraries were prepared from the 12 raw extracts using the TruSeq Nano DNA Low Throughput Library Prep Kit (Illumina, CA, USA) with TruSeq DNA Single Indexes Set A (Illumina). We followed the manufacturer’s protocol, except that we retained all DNA fragments by not removing large fragments and by adding 200 µL Sample Purification Beads (instead of 30 µL as per Illumina protocol) in the “small fragments removal” step. Instead of purifying our libraries using magnetic beads we ran them on a 1.5% agarose gel and cut out bands between 200 - 300 bp using sterile scalpels. We pooled the gel pieces of our duplicate libraries in one vial and purified them using the NucleoSpin Gel and PCR Clean-up kit and protocol (Macherey Nagel, Germany). We washed and eluted the DNA twice with the same 12 µL elution buffer and quantified the libraries using the Qubit dsDNA HS Assay (Invitrogen, MA, USA). The DNA-content of library of sample 41/42 (4.35 mbsf) was very low, thus we added an ethanol precipitation step (final volume 6 µL), and then pooled the barcoded libraries into an equimolar 10 nM pool (except for sample 41/42, 4.35 mbsf, which was 7.64 nM). The samples were sequenced on a HiSeq 4000 (2 ×150 bp cycle; ~350 Mio paired-end reads total, i.e., ~58Mio/sample, and an approximate sequencing depth of 20X/sample assuming a diatom genome-size of 80–100 Mb) at Fasteris, Switzerland.
3
Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted from thoracic muscle tissue using the DNeasy Blood and Tissue Kit (QIAGEN). The extracted genomic DNA was processed using the TruSeq Nano DNA Low Throughput Library Prep Kit (Illumina, Inc.). The resulting libraries were outsourced to Macrogen Japan for sequencing, which was conducted using a HiSeq X Ten Sequencing System (Illumina Inc.; 150‐bp paired ends). A minimum of 6 Gbp were obtained per individual, which corresponds to approximately 29× coverage in terms of genome size for this subspecies (211 Mbp; Yokoi et al., 2018 ). The original NGS reads were registered with the DNA Data Bank of Japan database (under BioProject PRJDB14080, DRR397616‐DRR397720).
4
Whole-Genome Sequencing of Probiotic Bacteria
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Bacteria for use in the study were selected based on probiotic characteristics, and whole-genome sequencing was performed. Total DNA of the selected strain was extracted using the NucleoSpin Microbial DNA Mini kit (Macherey–Nagel, Germany) according to the manufacturer's instructions. The extracted DNA yield was assessed using the Qubit with High sensitivity range kit (Thermo Fisher Scientific, USA). The sequencing library for genomic DNA was prepared using the TruSeq Nano DNA Low Throughput Library Prep kit (Illumina, USA). Sequencing was carried out using the MiSeq illumine system. After sequencing, individual sequence reads were checked for quality control using FastQC v.0.11.8. Illumina Sequencing data were processed to remove low-quality bases and adapter sequences with optimized settings (LEADING, 10; TRAILING, 10; SLIDINGWINDOW, 4:20; MINLEN, 200) using Trimmomatic v0.39. Subsequently, additional phiX control,s were removed from preassembled data. Trimmed sequences were aligned against the phiX genome using bowtie2 v2.3.5.1 with the default options and filtered out using samtools v1.9. Each of these assembled contigs was annotated using the PATRIC and RAST bacterial databases, and the coding sequence, ribosomal RNA and transfer RNA from their contig sequences were identified.
5
Bisulfite Sequencing Library Preparation
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E14TG2a gDNA was precleaned with Genomic DNA Clean and Concentrator kit (ZR). 500 ng of DNA in each reaction was BS converted with EZ DNA Methylation-Gold Kit (ZR) using standard protocol and purified DNA was eluted with 10 μl of M-Elution Buffer. Selected regions were then amplified for 25 cycles with Phusion U HS polymerase (TS) using primers specific to each strand (Table S2 ) and DNA was purified with DNA clean and concentrator kit (ZR). Fragments were PAGE-purified using SYBR Gold staining (Invitrogen), crushed gel was incubated in an elution buffer (0.5 M CH3COONH4, 0.1 mM EDTA, 0.1% SDS) for 2 h at 37°C, shaking at 600 rpm. Samples were filtered through Costar Spin-X (Corning) centrifuge filters and DNA was purified using DNA clean and concentrator kit (ZR). DNA was end-repaired, then 3′-dA mononucleotide extension was added to the end-repaired DNA as described in Gibas et al.18 and barcoded Illumina adapters (from TruSeq Nano DNA Low Throughput Library Prep Kit, Illumina) were ligated. After 4 cycles of amplification with Illumina specific primers and Platinum SuperFi PCR Master Mix (TS), DNA was purified using GeneJET NGS Cleanup Kit (TS). Libraries were tested on Agilent 2100 Bioanalyzer (Agilent Technologies) and subjected to Illumina sequencing (MiniSeq, DNA Sequencing Center, VU Life Sciences Center, Lithuania).
6
Library Construction Methods for CNV-seq
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An overview of the three library construction methods used in this study is shown in Figure 1 . All genomic samples for library construction were quantified using Qubit 3.0 (Invitrogen, Waltham, MA, USA). For PCR-free-frag library construction used by rCNV-seq (Method 1), gDNA (10–40 ng) was initially treated by dsDNA fragmentase at 37 °C (NEBNext dsDNA Fragmentase, New England Biolabs, Ipswich, MA, USA) to produce smaller derivatives with an average size of ~200 bps. Prepared gDNA fragments were then end-repaired, A-tailed and then ligated with barcoded sequencing adaptors using a proprietary DNA repair kit (KR2000, Berry Genomics, Beijing, China) to generate libraries for sequencing. For PCR-free-soni (Method 2) and PCR-soni (Method 3) used for commercial CNV-seq, gDNA (1 µg, PCR-free-soni; 100–200 ng PCR-soni) was sheared by sonification and fragments of 350 bps size selected on agarose gels using TruSeq DNA PCR-Free Low Throughput Library Prep Kit (Illumina, San Diego, CA, USA) and TruSeq Nano DNA Low Throughput Library Prep Kit (Illumina, San Diego, CA, USA), respectively (Figure 1 ).
7
Whole Genome Sequencing of S. Infantis
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Library preparation was done using TruSeq Nano DNA Low Throughput Library Prep Kit (Illumina, San Diego, CA, USA). Whole genome sequencing was performed as pairedend (150 bp × 2) sequencing on Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA). The aim of coverage was > 30 × . FastQ file was uploaded to the Galaxy platform (https ://usega laxy.org/) and the sequences of adaptors and barcodes were removed. Mapping of the sequences was done using Geneious Prime 11.0.5 (www.genei ous.com) software. The genome sequences of the wild-type and mutant S. Infantis strains were submitted to the GenBank under the access numbers CP040600 and CP040601, respectively.
8
Phage DNA Extraction and Library Preparation
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The purified phage sample (300 µL) was treated with 50 μg of proteinase K (20 mg/mL; ThermoFisher, Wlatham, MA, USA) and with an addition of SDS (Sigma-Aldrich) up to a 0.5% of final concentration before being incubated for an hour at +56 °C and subjected to DNA extraction using Genomic DNA Clean & Concentrator-10 (Zymo Research, Irvine, CA, USA) according to the manufacturer’s guidelines.
To prepare the input for the NGS library, 200 ng of phage DNA was randomly physically sheared using Covaris S220 focused-ultrasonicator (Covaris, Woburn, MA, USA) with a target fragment length of 550 bp. Fragmented DNA was used as an input for barcoded TruSeq DNA Nano Low Throughput Library Prep Kit (Illumina, San Diego, CA, USA) as per the manufacturer’s reference guide using adapter 7 from TruSeq DNA Single Indexes Set A (Illumina). The quality and quantity of the final library were verified using an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) with a high Sensitivity DNA kit (Agilent) and Qubit fluorometer (Invitrogen, Waltham, MA, USA) dsDNA high-sensitivity quantification assay (Invitrogen). Library sequencing was carried out on the Illumina MiSeq system (Illumina) using a 500-cycle MiSeq Reagent Kit v2 nano (Illumina) as one of the 12 pooled differently barcoded libraries.
To prepare the input for the NGS library, 200 ng of phage DNA was randomly physically sheared using Covaris S220 focused-ultrasonicator (Covaris, Woburn, MA, USA) with a target fragment length of 550 bp. Fragmented DNA was used as an input for barcoded TruSeq DNA Nano Low Throughput Library Prep Kit (Illumina, San Diego, CA, USA) as per the manufacturer’s reference guide using adapter 7 from TruSeq DNA Single Indexes Set A (Illumina). The quality and quantity of the final library were verified using an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) with a high Sensitivity DNA kit (Agilent) and Qubit fluorometer (Invitrogen, Waltham, MA, USA) dsDNA high-sensitivity quantification assay (Invitrogen). Library sequencing was carried out on the Illumina MiSeq system (Illumina) using a 500-cycle MiSeq Reagent Kit v2 nano (Illumina) as one of the 12 pooled differently barcoded libraries.
9
Covaris Shearing and Illumina Sequencing
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Each pool was sheared on a Covaris M220 (Covaris, Woburn, MA, USA) in a 130 μL Covaris Adaptive Focused Acoustics (AFA) microtube (Covaris, Woburn, MA, USA) to a target size of ~350–400 bp, with the following settings: 50 W peak incident power, 20% duty factor, 200 cycles per burst, 65 s treatment time.
Successful fragmentation was checked by running 1 μL of the sheared pool with a Bioanalyzer High-Sensitivity DNA kit on a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA).
The validated pools were then processed with the TruSeq DNA Nano Low-Throughput Library Prep kit and TruSeq DNA Single Index Set A and B, according to the manufacturer’s protocol (Illumina, San Diego, CA, USA).
Molarities of the libraries were calculated and diluted to a 4 nM working concentration. The different libraries that were to be sequenced together (with different indexes) were pooled proportionally to their total genomic target size. Subsequently, these pools were denatured with 0.2N NaOH and loaded into a MiSeq Reagent Kit V2 (300 cycles) cartridge, according to the manufacturer’s recommendations (Illumina, San Diego, CA, USA). Paired-end sequencing (2× 151 cycles) was performed on a MiSeq instrument (Illumina, San Diego, CA, USA).
A detailed step-by-step protocol is provided in theSupplementary Materials (Text S1 ).
Successful fragmentation was checked by running 1 μL of the sheared pool with a Bioanalyzer High-Sensitivity DNA kit on a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA).
The validated pools were then processed with the TruSeq DNA Nano Low-Throughput Library Prep kit and TruSeq DNA Single Index Set A and B, according to the manufacturer’s protocol (Illumina, San Diego, CA, USA).
Molarities of the libraries were calculated and diluted to a 4 nM working concentration. The different libraries that were to be sequenced together (with different indexes) were pooled proportionally to their total genomic target size. Subsequently, these pools were denatured with 0.2N NaOH and loaded into a MiSeq Reagent Kit V2 (300 cycles) cartridge, according to the manufacturer’s recommendations (Illumina, San Diego, CA, USA). Paired-end sequencing (2× 151 cycles) was performed on a MiSeq instrument (Illumina, San Diego, CA, USA).
A detailed step-by-step protocol is provided in the
10
Covaris-based DNA Fragmentation and Library Prep
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Each pool was sheared on a Covaris M220 (Covaris, Woburn, USA) in a 130 μl Covaris AFA microtube (Covaris, Woburn, USA) to a target size of ∼ 350-400 bp, with the following settings: 50 W peak incident power, 20% duty factor, 200 cycles per burst, 65 s treatment time.
Successful fragmentation was checked by running 1 μl of the sheared pool with a Bioanalyzer High Sensitivity DNA kit on a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, USA).
The validated pools were then processed with the TruSeq DNA Nano Low Throughput Library Prep kit and TruSeq DNA Single Index Set A and B, according to the manufacturer's protocol (Illumina, San Diego, USA).
Molarities of the libraries were calculated and diluted to a 4 nM working concentration. The different libraries that were to be sequenced together (with different indexes) were pooled proportionally to their total genomic target size.
Subsequently, these pools were denatured with 0.2N NaOH and loaded into a MiSeq Reagent Kit V2 (300-cycles) cartridge, according to the manufacturer's recommendations (Illumina, San Diego, USA). Paired-end sequencing (2x 151 cycles) was performed on a MiSeq instrument (Illumina, San Diego, USA).
A detailed step-by-step protocol is provided in the Supplementary Materials (Text S1).
Successful fragmentation was checked by running 1 μl of the sheared pool with a Bioanalyzer High Sensitivity DNA kit on a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, USA).
The validated pools were then processed with the TruSeq DNA Nano Low Throughput Library Prep kit and TruSeq DNA Single Index Set A and B, according to the manufacturer's protocol (Illumina, San Diego, USA).
Molarities of the libraries were calculated and diluted to a 4 nM working concentration. The different libraries that were to be sequenced together (with different indexes) were pooled proportionally to their total genomic target size.
Subsequently, these pools were denatured with 0.2N NaOH and loaded into a MiSeq Reagent Kit V2 (300-cycles) cartridge, according to the manufacturer's recommendations (Illumina, San Diego, USA). Paired-end sequencing (2x 151 cycles) was performed on a MiSeq instrument (Illumina, San Diego, USA).
A detailed step-by-step protocol is provided in the Supplementary Materials (Text S1).
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