The formalin-fixed paraffin-embedded (FFPE) DNA was extracted using the MagPure FFPE DNA LQ Kit following the manufacturer’s instruction. The sample’s concentration was detected by Qubit fluorometer. The integrity and purification of samples were detected by agarose gel electrophoresis. One nanogram and more FFPE DNA were randomly fragmented by Covaris. Fragmented DNAs were tested by Agilent 2100 and purified by the Agencourt AMPure XP kit. The selected fragments were end-repair and 3′ adenylated; at the same time, the adapters were ligated to the ends of these 3′ adenylated fragments. These fragments were amplified with KAPA HiFi HotStart DNA polymerase, and the PCR products were purified with the Agencourt AMPure XP kit. The library was qualified by the Agilent 2100 Bioanalyzer and ABI StepOnePlus real-time PCR (RT-PCR) system. The qualified libraries were sequenced pair end on the Hiseq4000/Xten/Novaseq system (BGI, Shenzhen, China). We used Genome Analysis Toolkit software to detect (Single Nucleotide Variants) SNVs and indels. All SNVs and indels were filtered and estimated via multiple databases, including Genome Aggregation Database and Exome Aggregation Consortium. Common variants and rare variants were classified according to minor allele frequencies (MAFs; common variants: MAF ≥0.01, rare variants: MAF <0.01).14 (link)
Ampure xp kit
Manufactured by Beckman Coulter
Sourced in United States, United Kingdom, China
The AMPure XP kit is a magnetic bead-based nucleic acid purification system. It is designed to selectively bind DNA or RNA molecules, allowing for efficient purification and recovery from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Aligner version 3, by CodonCode (21 mentions)
Nextseq 500 sequencer, by Illumina (12 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions)
2100 bioanalyzer, by Agilent Technologies (8 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (8 mentions)
Taq dna polymerase, by Qiagen (7 mentions)
Qiaamp dna mini kit, by Qiagen (7 mentions)
Nextseq 500, by Illumina (7 mentions)
Hiseq 2500, by Illumina (6 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
3730xl dna analyzer 96 capillary sequencer, by Thermo Fisher Scientific (5 mentions)
Dneasy plant mini kit, by Qiagen (4 mentions)
Nextseq 500 platform, by Illumina (4 mentions)
M220 focused ultrasonicator, by Covaris (4 mentions)
Hiseq 2000, by Illumina (4 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (4 mentions)
Miseq platform, by Illumina (4 mentions)
Mastercycler gradient, by Eppendorf (4 mentions)
Quant it picogreen dna assay, by Thermo Fisher Scientific (4 mentions)
Pe9700 thermal cycler, by PerkinElmer (3 mentions)
123 protocols using ampure xp kit
1
FFPE DNA Sequencing Library Preparation
2
DNA Extraction and Sequencing from Dried Voucher Specimen
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Genomic DNA was isolated from 10 mg of a dried voucher specimen (JBSD 127410), using the DNeasy Plant Mini Kit (Qiagen, Milan) according to the manufacturer’s instructions. Primers LR0R/LR6 (Vilgalys and Hester 1990 (link), Vilgalys lab. http://www.botany.duke.edu/fungi/mycolab ) were used for the nrLSU (28S) DNA amplification and universal primers ITS1F/ITS4 for the ITS region amplification (White et al. 1990 , Gardes and Bruns 1993 (link)). Amplification reactions were performed in a PE9700 thermal cycler (Perkin-Elmer, Applied Biosystems, Norwalk) in 25 ml reaction mixtures using the following final concentrations or total amounts: 5 ng DNA, 1 × PCR buffer (20 mM Tris/HCl pH 8.4, 50 mM KCl), 1 mM of each primer, 2.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 unit of Taq polymerase (Promega, Madison). The PCR programme was as follows: 3 min at 95 °C for 1 cycle; 30 s at 94 °C, 45 s at 50 °C, 2 min at 72 °C for 35 cycles, 10 min at 72 °C for 1 cycle. PCR products were resolved on a 1% agarose gel and visualised by staining with ethidium bromide. The PCR products were purified with the AMPure XP kit (Beckman Coulter, Pasadena) and sequenced by MACROGEN (Seoul). The sequences were submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank/ ) and their accession numbers are reported in Figs 1 –2 .
3
ChIP-Seq Library Preparation and Analysis
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Sequencing libraries were prepared from 1.0 ng of DNA subjected to ChIP and input samples by a Mondrian SP+ system (Nugen) with an Ovation SP ultralow DR multiplex system (Nugen). The libraries were further purified and size-selected using an AMPure XP kit (Beckman Coulter) and were quantified by a quantitative MiSeq (qMiSeq) method (65 (link)). The samples were sequenced on a HiSeq 2500 (Illumina) that generated 101-base reads. Sequencing data were aligned with the hg19 reference genome with Bowtie2 (66 (link)), and peaks were called with MACS2 (67 (link)). ChIP-seq peak visualization was done with Integrative Genomic Viewer (68 (link)).
4
Genomic DNA Sequencing Preparation
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Genomic DNA from SKU1108 for genome sequencing was prepared as previously reported (23 (link)). Genomic DNA (~20 μg) was purified with the AMPure Xp Kit (Beckman Coulter, Beverly, MA, USA). DNA was sheared using a Covaris g-TUBE (Covaris, Woburn, MA, USA) following the manufacturer’s recommendations for 20-kb fragments. A sequencing library was prepared using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences of California, Menlo Park, CA, USA). Libraries for 2 SMRT cells (~10 μg) were constructed using two different methods. Small library fragments were removed using BluePippin from one SMRT cell (Sage Science, Beverly, MA, USA) with a 10-kb cut-off. In the other cell, small library fragments were not removed.
5
Mitochondrial DNA Barcoding Protocol
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Total genomic DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions. The 5′ region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers TelF1 (5′–TCGACTAATCAYAAAGAYATYGGCAC–3′) and TelR1 (5′–ACTTCTGGGTGNCCAAARAATCARAA–3′) [21 ]. PCR reactions were performed in 20 μL, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94 °C, followed by 40 cycles at 94 °C for 30 s, 48 °C for 40 s, and 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified (Ampure XP Kit, Beckman Coulter, Brea, CA, USA) and sequenced in both directions on a 3730 × l DNA Analyzer 96-capillary sequencer (Applied Biosystems, Foster City, CA, USA). We used CodonCode Aligner version 3.7.1 software (Codon Code Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank (accession numbers in Table 1 ). Species identification was confirmed with the BOLD identification engine [67 (link)].
6
16S Metagenomic Library Preparation and Sequencing
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After evaluating the amplified products on 2% w/v agarose gel, the products were purified using Ampure XP kit (Beckman Coulter, Brea, CA, United States). The libraries were prepared using Illumina 16S metagenomic library preparation guide and evaluated on 2100 Bioanalyzer using Bioanalyzer DNA 1000 kit (Agilent, United States) to estimate the library size and Qubit 2.0 flourometer using Qubit dsDNA HS kit (Life technologies, United States) to estimate the library concentration. After this, 150 bp paired-end sequencing of both the libraries was performed on the Illumina NextSeq 500 platform (Illumina, United States) using NextSeq 500/550 v2 sequencing reagent kit (Illumina Inc., United States) at the Next-Generation Sequencing (NGS) Facility, Indian Institute of Science Education and Research (IISER) Bhopal, India.
7
16S rRNA Gene Amplification and Sequencing
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The V4 region of 16S rRNA gene was amplified by PCR with forward and reverse primers according to Kozich et al. (28 (link)), containing Illumina (Illumina Inc., San Diego, CA, USA) adapter sequences and unique dual indexes used to tag each PCR product, according to the 16S-protocol provided by Illumina. Primer sequences can be found in Table 1 . Briefly, PCR was carried out in 25 μL reactions with 0.2 μM forward and reverse primers, with 12.5 ng template DNA and 12.5 μL of 2 × KAPA HiFi HotStart Ready Mix kit (KAPA Biosystems, Woburn, MA, USA). Thermal cycling consisted of initial denaturation at 95°C for 3 min followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, followed by a final step of 72°C for 5 min. The amplicon products were purified with Agencourt AmPureXP Kit (Beckman Coulter, Miami, FL, USA). Next, a second PCR was thereafter performed to attach Illumina adapters and unique dual indexes to each sample, followed by a clean-up step with AmPureXP Kit (Beckman Coulter). PCR amplicons were visualized using 0.1% agarose gel electrophoresis to verify the size of the amplicon. Negative extraction controls did not produce visible bands.
8
Metagenomic 16S rRNA Gene Sequencing
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The extracted DNA (5 ng) was PCR amplified with seven different custom modified 5ʹ-end adaptor-ligated 341F and 534R primers (see the Primer Details section in Additional File 1) targeting the V3 hypervariable region of 16S rRNA gene. After evaluating the amplified products on 2% w/v agarose gel, the products were purified using Ampure XP kit (Beckman Coulter, Brea, CA). Amplicon libraries were prepared by following the Illumina 16S rRNA gene metagenomic library preparation guide. Metagenomic libraries were prepared using Illumina Nextera XT sample preparation kit (Illumina Inc.) by following the manufacturer's protocol. Library size of all the libraries was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and quantified on a Qubit 2.0 fluorometer using Qubit dsDNA HS kit (Life technologies) and by qPCR using KAPA SYBR FAST qPCR Master mix and Illumina standards and primer premix (KAPA Biosystems, Wilmington, MA) following the Illumina suggested protocol. Both the amplicon and metagenomic libraries were loaded on Illumina NextSeq 500 platform using NextSeq 500/550 v2 sequencing reagent kit (Illumina Inc.), and 150 bp paired-end sequencing was performed at the Next-Generation Sequencing (NGS) Facility, IISER Bhopal, India.
9
Genomic DNA Extraction and Sequencing from Tissue Samples
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Using the DNeasy Blood and Tissue kit (Qiagen) and QIAamp DNA FFPE Tissue Kit
(Qiagen) in accordance with the manufacturer's instructions, genomic DNA was
extracted from fresh tissue and FFPE samples.29 (link) Tissue DNA was sheared
using Covaris M220 (Covaris, MA, USA), followed by end repair, phosphorylation
and adaptor ligation. Fragments between 200 and 400 bp from the sheared tissue
DNA were purified (Agencourt AMPure XP Kit, Beckman Coulter, CA, USA), followed
by hybridization with capture probes baits, hybrid selection with magnetic
beads, and PCR amplification. The quality and the size of the fragments were
assessed by high sensitivity DNA kit using Bioanalyzer 2100 (Agilent
Technologies, CA, USA). Indexed samples were sequenced on Nextseq500 (Illumina,
Inc., USA) with paired-end reads and target sequencing depth of 1,000× for
tissue samples.
(Qiagen) in accordance with the manufacturer's instructions, genomic DNA was
extracted from fresh tissue and FFPE samples.29 (link) Tissue DNA was sheared
using Covaris M220 (Covaris, MA, USA), followed by end repair, phosphorylation
and adaptor ligation. Fragments between 200 and 400 bp from the sheared tissue
DNA were purified (Agencourt AMPure XP Kit, Beckman Coulter, CA, USA), followed
by hybridization with capture probes baits, hybrid selection with magnetic
beads, and PCR amplification. The quality and the size of the fragments were
assessed by high sensitivity DNA kit using Bioanalyzer 2100 (Agilent
Technologies, CA, USA). Indexed samples were sequenced on Nextseq500 (Illumina,
Inc., USA) with paired-end reads and target sequencing depth of 1,000× for
tissue samples.
10
DNA Fragmentation and Target Enrichment
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DNA fragmentation was performed using a Covaris M220 Focused‐ultrasonicator (Woburn, MA, USA), followed by end repair, phosphorylation, and adaptor ligation. Fragments of 200–400 bp were selected using AMPure beads (Agencourt AMPure XP Kit, Beckman Coulter, CA, USA), followed by hybridization with capture probe baits, hybrid selection with magnetic beads, and PCR amplification. Subsequently, high‐sensitivity DNA assay was performed to assess the quality and size of all fragments.
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