Ultra dna library prep kit

One hundred ng of gDNA was sonicated to a fragment size of 500 bp using a Covaris ultrasonicator and processed using NEB Ultra DNA library prep kit according to the manufacturer’s instructions. The constructed library was quantified and size-estimated using Qubit and Tapestation2100, respectively. Sequencing of the library was performed on an Illumina MiSeq located at the Monash University Malaysia Genomics Facility using the run configuration 2 × 250 bp.

The target region for sequencing of the candidate breast cancer susceptibility genes were selected based on coding exons. For established genes, intragenic regions were also included. Baits for solution based hybrid selection capture^{32 (link)} were designed using the Agilent SureSelect design tool (https://earray.chem.agilent.com/suredesign/). 500 nanograms of high molecular weight DNA was fragmented using a Covaris E-220. Fragmentation was verified by bioanalyzer. Library preparation was performed using the NEBNext Ultra DNA Library Prep kit (E7370L), and NEB Dual indexed adapters. Fragmented DNA (mean fragment size 300 ± 20 bp) was end repaired, adapter ligated, and size selected using Ampure XP /SPRI (A63881) and was PCR amplified for eight cycles. A post-PCR clean-up was performed and enrichment for targets was performed using the Agilent SureSelect protocol. Yields were assessed using BioAnalyzer. The mean library size was 300 ± 10 bp. NGS was performed on an Illumina HiSeq 2000 to an estimated 100× mean coverage for the target region to yield paired-end reads of 100 bp per sample, using 24 samples/lane.

The 15-kb SMRTbell library was constructed using the SMRTbell Express Template Prep Kit (version 2.0). The 350-bp small, fragmented library was constructed using the NEBNext ultra DNA library prep kit. After the library was qualified, the whole genome of D. rubrovolvata Di001 was sequenced using the PacBio Sequel II platform and Illumina NovaSeq PE150 at the Biomarker Technologies Corporation (Beijing, China), and the sequencing results were used for gene annotation.

We isolated total DNA from rat fecal pellets using the cetyl trimethyl ammonium bromide/sodium dodecyl sulfate (CTAB/SDS) method. We selected the V3–V4 region of 16S rRNA genes using specific primers with the following barcode: 341 F-806R. The polymerase chain reaction (PCR) products were detected by 2% agarose gel electrophoresis and the target fragments were cut and recovered. The AxyPrepDNA (Axygen Scientific Inc. USA) gel recovery kit was used.
We detected the PCR amplification products by the QuantiFluor™-ST blue fluorescence quantitative system (Promega Corporation, Madison, WI, USA). The corresponding proportions were mixed according to sequencing volume requirements of each sample. Subsequently, we used the Ultra™ DNA Library Prep Kit (NEB, USA) for library construction. The library was sequenced on a computer following quality-check performed by agilent bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) and qubit.
We performed sequence analyses using the UPARSE software package with the UPARSE-OTU and UPARSE-out ref algorithms. In-house perl scripts were used to analyze the alpha (within samples) and beta (among samples) diversity values. We assigned sequences with in-house perl to the same operational taxonomic units (OTUs).

DNA was extracted from leaf tissue using a Qiagen DNeasy Plant Mini Kit, and a 270-bp paired-end library was generated using an NEBNext Ultra DNA Library Prep Kit. Sequencing was performed using Illumina HiSeq2500 and Hiseq4000 platforms. In total, 30.65 Gb (~66×) of data were obtained, and the reads were trimmed using Trimmomatic^{30 (link)} (v.0.36) with default parameters.

Weissella confusa LM1 was cultured in MRS broth at 37°C for 12 h. Genomic DNA was extracted by using Qiagen DNeasy Blood & Tissue Kit (Qiagen) according to the instructions of the manufacturer. Harvested DNA quality was checked by agarose gel electrophoresis (0.8%), and DNA quantity was determined by using NanoDrop 1000 (Thermo Fisher Scientific). The whole-genome sequence of W. confusa LM1 was obtained through single-molecule real-time (SMRT) sequencing by using PacBio Sequel platform and through next-generation sequencing (NGS) by using Illumina NovaSeq platform. Libraries for SMRT sequencing were constructed with an insert size of 10 kb by using the SMRTbell Template kit (Pacific Biosciences). Libraries for NGS were constructed with an insert size of 350 bp by using Ultra DNA Library Prep Kit (NEB).