The A3B and empty plasmid containing strains were streaked out on complete media with hygromycin. 12 to 24 individual colonies were plated on media lacking tryptophan. The colonies were also inoculated in YPD overnight for DNA extraction (Trp− isolates). Genomic DNA was also extracted for the Trp+ revertants obtained from each isolate. DNA libraries were prepared using the KAPA hyper kit (KAPA Biosystems, Wilmington, MA), and paired end 100 bp or125 bp reads from the HiSeq 2500 sequencer or 150 bp reads from the HiSeq 4000 sequencer (Illumina, San Diego, CA). The mutation analysis was done using the CLC-genomics workbench (Qiagen, Redwood City, CA) as follows. The sequences were aligned to the reference genome from ySR128 [18 (link)] using default parameters, and were realigned using variants detected from the “low frequency variant detection” tool. Duplicate mapped reads were removed and the “fixed ploidy variant detection” tool was used with ploidy annotated as 1, and required variant probability of 90%. Only homozygous mutations were considered. To remove mutation calls accrued in the parental strains, the variants in each Trp+ isolate were compared against the parental Trp− isolates. Only base substitutions that were present in the Trp+ strains but not in the parent strains, were analyzed further.
Kapa hyper kit
Manufactured by Roche
Sourced in United States
The KAPA Hyper kit is a reagent kit designed for highly efficient and rapid DNA amplification. The kit contains enzymes, buffers, and other components optimized for high-throughput and sensitive PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000 platform, by Illumina (2 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
Hiseq 4000 sequencer, by Illumina (1 mentions)
Clc genomics workbench, by Qiagen (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Dneasy powersoil kit, by Qiagen (1 mentions)
Qubit spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Miseq high throughput sequencing, by Illumina (1 mentions)
Focused ultrasonicator, by Covaris (1 mentions)
Rnaeasy plus kit, by Qiagen (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Miseq reporter software, by Illumina (1 mentions)
D1000 tapestation tape, by Agilent Technologies (1 mentions)
Hyper library preparation kit, by Roche (1 mentions)
Nanodrop system, by Thermo Fisher Scientific (1 mentions)
Allprep powerviral dna rna kit, by Qiagen (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
12 protocols using kapa hyper kit
1
Genomic Analysis of Tryptophan Revertants
2
Single-Cell Bisulfite-Free DNA Methylation
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mESCs gDNA was spiked with 0.5% of methylated lambda DNA, 0.025% of 2-kb-unmodified and 0.025% of 2-kb-caC spike-in controls. For CAPS approach, gDNA was fragmented by Covaris M220 instrument and size-selected to 200–400 bp using Ampure XP beads (Beckman Coulter). For other approaches, gDNA was fragmented and size-selected to 300–500 bp; 0.01% of synthetic oligo with N5mCNN/N5hmCNN sequences and 0.01% of synthetic oligo with 5fC modifications were added after size-selection. One-hundred nanograms of fragmented DNA was used for end-repair/A-tailing and ligation of NEBNext Adaptor (NEB) with KAPA Hyper kit (KAPA) according to the manufacturer’s protocol. The uracil in the loop of NEBNext Adaptor was removed by adding 3 μL of USER enzyme (NEB) to the ligation reaction and incubating for 15 min at 37 °C. Then the reaction was purified with 0.8× Ampure XP beads according to the manufacturer’s protocol. For CAPS approach, 80% acetonitrile:H2O was used instead of 80% ethanol:H2O during the beads purification step.
3
Fecal Metagenomics Sequencing Protocol
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Metagenomics sequencing was performed at the Alkek Center for Metagenomics and Microbiome Research at Baylor College of Medicine. Briefly, total DNA was extracted from fecal samples using a DNeasy PowerSoil Kit (Qiagen, Germantown, MD, USA) followed by library construction using a KAPA Hyper kit (Kapa Biosystems, Wilmington, MA, USA). The NovaSeq 6000 platform was used for the metagenomics sequencing (Illumina, San Diego, CA, USA) with 2×150 bp paired-end read protocol.
4
Optimized gDNA Fragmentation and Amplification
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Example 3
Total gDNA from a sample was Covaris fragmented to approximately 150 bp. The fragments were end-repaired with an end-repair and A-tailing kite (e.g., KAPA® Hyper kit; Wilmington, Mass.). The result was a plurality of dA tailed gDNA fragments. These gDNA fragments were then incubated with a universal adapter polynucleotide in the presence DNA ligase, followed by polymerase chain reaction (PCR) amplification with a forward primer complementary to the universal adapter sequence and a plurality of 22 different reverse primers complementary to a variety of sequences within the gDNA.
All 22 primer sets resulted in amplicons with anticipated multiple product sizes from the ligated templates.
5
Optimized gDNA Fragmentation and Amplification
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Example 3
Total gDNA from a sample was Covaris fragmented to approximately 150 bp. The fragments were end-repaired with an end-repair and A-tailing kite (e.g., KAPA® Hyper kit; Wilmington, Mass.). The result was a plurality of dA tailed gDNA fragments. These gDNA fragments were then incubated with a universal adapter polynucleotide in the presence DNA ligase, followed by polymerase chain reaction (PCR) amplification with a forward primer complementary to the universal adapter sequence and a plurality of 22 different reverse primers complementary to a variety of sequences within the gDNA.
All 22 primer sets resulted in amplicons with anticipated multiple product sizes from the ligated templates.
6
DNA Sequencing Library Preparation
7
Metagenome cDNA Library Preparation and Sequencing
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The transcribed cDNA obtained from the natural bacterial community was fragmented (~500 bp) with a Covaris focused‐ultrasonicator. The concentration of the obtained cDNA fragments was measured with a QUBIT spectrophotometer (ThermoFisher) following the manufacturer's protocol. The library preparation was performed with a KAPA Hyper kit (KAPA Biosystems). Briefly, 10 ng of cDNA were end repaired and A‐tailed. Subsequently, the genetic material was ligated and purified using a bead‐based cleanup method. The obtained libraries were PCR amplified with following conditions: 98 °C for 45 s, 12 cycles of denaturation at 98 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 1 min and hold at 4 °C. The PCR products were further purified using a bead‐based cleanup method. The libraries concentrations were measured by qPCR (KAPA Hyper kit) following the manufacturer's recommendations, and 2.5 nM cDNA of each sample was sequenced using an Illumina MiSeq high throughput sequencing (2 × 250 paired‐end platform) at JAMSTEC, Japan. The sequence data generated are publically available in the DDBJ sequence read archive (DRA) under the accession number DRA008144 for sample t0, DRA008145 for sample t5, DRA008146 for sample t40 and DRA008147 for sample t60.
8
RNA-Seq and Barcoded Gene Expression Analysis
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The F3 hiPSC line was utilized for the RNA-Seq and barcode capture experiment as follows:
Cells were nucleofected as described above with 100fmol of a barcoded cDNA vector harboring sfGFP or the gene ZGLP1 and 24 fmol of super piggyBac transposase in duplicate.
Integrants were purified through puromycin drug selection as detailed above
The cells were then induced under 1 μg/mL doxycycline for 3 days and RNA was harvested using the Qiagen RNAeasy plus kit.
RIN scores were determined using a Bioanalyzer and only samples with RIN score greater than 8 were utilized.
An RNA-Seq cDNA library was constructed using the KAPA Hyper Kit with RiboErase
RNA-Sequencing was performed on the Next-Seq 500 Illumina platform.
Reads were processed and aligned to the hg19 build using STAR Aligner
DEGs were determined using DESeq2.
Gene barcodes were identified using BBMap.
DESeq2 results for gene expression log2fc and their matching p values were plotted utilizing the R package ggplot2.
9
Optimized Canine Whole Exome Sequencing
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Pre-capture libraries were prepared with standard library preparation protocols using the KAPA Hyper kit from Kapa Biosystems and then pooled at equal volume and sequenced on the Illumina platform at low depth to determine exact relative abundances. Based on these abundances, libraries were balanced optimally for whole exome sequencing using the SureSelect Canine All Exon V2 bait set from Agilent. Library sequencing was performed on the Illumina Hiseq 2500 platform.
10
Low-Pass Whole-Genome Sequencing of FFPE Samples
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sWGS, also known as low-pass whole-genome sequencing, was performed on DNA extracted from FFPE tumor samples (Maxwell 16 FFPE Plus LEV DNA Purification Kit, Promega, Madison, Wisconsin, USA). In addition, genomic DNA was extracted from whole blood using the DNeasy blood and tissue kit (Qiagen) to be used as reference.
Libraries from FFPE tumor tissue and normal peripheral blood DNA were constructed using the KAPA hyper kit (Roche) and sequenced on a NovaSeq 6000 platform (Illumina, paired end, 2×150) at the National Genomic Analysis Center (CNAG, Barcelona, Spain) to a goal of 6× mean target coverage.
Libraries from FFPE tumor tissue and normal peripheral blood DNA were constructed using the KAPA hyper kit (Roche) and sequenced on a NovaSeq 6000 platform (Illumina, paired end, 2×150) at the National Genomic Analysis Center (CNAG, Barcelona, Spain) to a goal of 6× mean target coverage.
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