Agilent dna 1000 kit

For generation of the targeted amplicon libraries the Ion AmpliSeq™ Custom 3G-Panelv2 (275 bp; Life Technologies Corporation; Carlsbad, CA, USA) consisting of 82 primer pairs to target all exons of the MSH2 and RAD50 genes (RefSeq:NM_000251.2 and RefSeq: NM_005732.3, respectively) was used. Polymerase chain reaction (PCR) was performed according to the manufacturer’s recommendations with the Ion AmpliSeq™ Library Kit 2.0. Amplicon size distribution and library concentration was determined using Agilent DNA 1000 Kit (Agilent Technologies; Inc., Waldbronn, Germany). The final concentration of the sample pool was measured by Qubit dsDNA BR Assay Kit (Life Technologies Corporation; Carlsbad, CA, USA). Emulsion PCR and sequencing was performed on an Ion PGM Sequencer (Life Technologies Corporation; Carlsbad, CA, USA) using 318 Chips and the Ion PGM 200 Sequencing Kit according to the manufacturer’s instructions. The sequence reads were mapped to the haploid human reference genome (hg19) with Novoalign (Novocraft Technologies). SNVs and short insertions and deletions (indels) were called using GATK version 2.8. [32 (link)] Variant annotation was performed with Jannovar [33 (link)].

To prepare Illumina sequencing compatible libraries for shotgun sequencing, 500 ng metagenomic DNA was mechanically sheared to generate 450–600 bp fragments using a Covaris S220 instrument (Covaris, Inc). Fragmented metagenomic DNA was end-repaired and 3′-adenylated, ligated with Illumina adapters, and PCR enriched with Illumina sequencing indexes (barcodes) using the NEBNext Ultra DNA library prep kit for Illumina (Catalogue # E7370L,New England BioLabs Inc). The quality and quantity of all the DNA libraries were analyzed with an Agilent DNA 1000 Kit on the 2100 Bioanalyzer Instrument and Qubit. DNA libraries were pooled in equimolar concentration and were sequenced following manufacturer’s protocol by multiplexing on Illumina MiSeq using the v3-600 kit for 301 paired-end read length and included an additional 12 cycles for the index. The average sequencing depth was approximately 3 million paired end reads per sample or 1.03 Gbp per sample.

Total RNA was extracted from 40 tissue samples. RNA fragmentation, double-stranded cDNA synthesis and adaptor ligation were performed using the Truseq^TM RNA Sample Prep Kit-v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Library concentration and size were assayed using the Qubit^® dsDNA HS Assay Kit in a Qubit^® 2.0 Fluorometer (Invitrogen, Lidingo, Sweden) and the Agilent DNA1000 Kit on a 2100 tape station (Agilent Technologies, Nærum, Denmark). Each library was normalized to a final concentration of 100 nM. All 22 samples were grouped into pools and diluted to a final concentration of 10 nM. Libraries were sequenced on a genome analyzer equipped with a paired-end module (Illumina) at the Technion – Israel Institute of Technology (Haifa, Israel) to generate 100-bp paired-end reads.

Library control was checked using an Agilent DNA 1000 kit on a Bioanalyzer 2,100 platform (Agilent, United States) and quantified using a QubitTM ssDNA Assay Kit (Invitrogen, United States). Then libraries were subjected to multiplex sequencing on a DNBSEQ platform (MGI, Shenzhen, China) acording to a “paired-end 100 bp” strategy.

RNA from F1 mice (NS vs HAL; n = 6 each) was subjected to RNA-seq. The RNA integrity number was calculated by an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA) and an Agilent RNA 6000 Nano Kit (Agilent Technologies Inc.) (supplementary Table 1). Subsequently, 200 ng total RNA from each sample was used for RNA-seq library preparation. An RNA-seq library of each sample was created using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Indianapolis, IN, USA) according to the manufacturer’s protocol. The quality of the average size (340–380 bp) was validated using an Agilent DNA1000 kit and Agilent 2100 Bioanalyzer (Agilent Technologies Inc.). The amount was determined using qPCR with the Kapa Library Quantification Kit (Illumina). The MiSeq Reagent kit V3 on a MiSeq system was used for sequencing (Illumina) based on pair-end reads (75 bp) according to the manufacturer’s instructions. The running cycle was set to 150 cycles.

For the development of genomic libraries, the SureSelect Strand-Specific RNA Library Prep Kit for Illumina Multiplexed Sequencing was used (Agilent Technologies, Santa Clara, California, USA), following the manufacturer’s instructions. To establish the quality of the amplified product, the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) and Agilent DNA 1000 kit (Agilent Technologies) were used. The concentration of the library under a range of 200 to 400 bp peaks was monitored. The library construction was done with 200 bp inserts, 100 bp paired-end sequencing and 40 M reads per sample. Sequencing of the Musa acuminata transcript was performed in the Illumina Hi Scan SQ™ (Towne Center Drive, San Diego, California, USA).