Dna sample prep kit

Re-sequencing analysis was commissioned by Insilicogen (Yongin, Republic of Korea) and performed using an Illumine Novaseq 6000 platform. A library was constructed from DNA fragments with 151 bp paired ends read using a DNA Sample Prep Kit (Illumina) following the manufacturer’s instructions. An analysis pipeline for detecting mutations in the sequencing data for the entire genome was employed with an NF-Core/SAREK workflow [32 (link)]. The snpEff tool was used for genetic variation annotation and effect prediction, while the snpEff database was referenced to Glycine max var. Williams 82 [11 (link)]. The whole genome sequencing data of GWS-1887 were deposited in NCBI under the BioProject PRJNA915129.

The genomic DNA of C. clavata BAUA-2787 was extracted using a DNeasy Plant Mini Kit (QIAGEN, Manchester, UK) after 2 days of cultivation in 100 mL CM medium at 26°C and 130 rpm. The DNA libraries were prepared with a 5500 SOLiD Mate-Paired Library Kit and sequenced by a 5500xl SOLiD system (Life Technologies, Carlsbad, CA, USA). A Nextera DNA Sample Prep Kit was also used for the library preparation, and the libraries were sequenced using the MiSeq platform (Illumina, San Diego, CA, USA). The hybrid de novo genome assembly (Ikegami et al., 2015 (link)) was performed using the MiSeq and SOLiD short read data. Additionally, the protein-coding genes in the draft genome were predicted by a combination of the gene prediction programs, ALN (Gotoh, 2000 (link)) and GlimmerHMM (Majoros et al., 2004 (link)). NCBI BLAST (http://blast.ncbi.nlm.nih.gov/) and the Pfam protein family database (http://pfam.xfam.org/) were used for sequence homology and conserved domain searches, as appropriate.

Paired-ends sequencing libraries were prepared from L. tarentolae genomic DNA with the Nextera DNA sample prep kit and sequenced on an Illumina HiSeq platform with 101-nucleotides paired-ends reads. Sequence reads were aligned to the L. tarentolae Parrot TarII PacBio genome assembly [22 (link)] using bwa-mem [23 (link)]. Read duplicates were marked using Picard and GATK was applied for single nucleotide variants and small insertions or deletions discovery [24 (link)]. Copy numbers variations were derived from read depth coverage as described earlier [25 (link)].

We used a Gentra Puregene Blood Kit (Qiagen) to extract the genomic DNA from a whole blood sample according to the manufacturer’s instructions. Then we assessed the quality of DNA by electrophoresis on 1% agarose gel and the quantity of DNA by a BioDrop mLITE pectrophotometer (a total of 15 mg of DNA was quantified using the spectrophotometer). The sequencing included two platforms: Illumina (San Diego, CA) Hiseq4000 and Pacific Biosciences (Menlo Park, CA) PacBio RSII. We used Illumina DNA Sample Prep Kit to construct three paired-end libraries with insert size of 350 bp and SMRT bell library preparation protocol (10 SMRT cells were sequenced) to generated by Pacific Biosciences long reads (> 10 kb).

Total RNA from hCGp7 cells used for qPCR was subjected to RNA-seq (Novogene, Beijing, China). RNA qualities were checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). RNA integrity number values were 10.0 in all samples. The RNA was subjected to prepare libraries using Illumina TruSeq RNA and DNA Sample Prep Kits (Illumina, San Diego, CA, USA). Library qualities were confirmed using a Qubit 2.0 fluorometer (Life Technologies; Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer. RNA-seq was performed using Illumina NovaSeq 6000 (Illumina) to generate >6 GB raw data per sample. Raw data were recorded in a FASTQ format. The quality of the read was calculated as the arithmetic mean of its Phred quality scores. Then the reads as follows were discarded with adaptor contamination, when uncertain nucleotides constitute >10% of either read, or when low quality nucleotides (base quality < 20) constitute >50% of the read. The cleaned reads were used for subsequent analyses. The reads were mapped to a reference genome GRCm39 using TopHat2. The numbers of the reads and mapping efficiencies were summarized in Table 1. The expression levels of the transcripts were calculated as FPKM using HTSeq.

The B.suis 019 strain was obtained from the key laboratory of prevention and control of animal disease, Shihezi University. This bacteria had been originally isolated from the sperm of sheep in the province of Xinjiang in China in 1983, then processed by freeze-drying for long-term preservation. In this study, B.suis 019 was cultured at 37 °C for 72 h using the streak plate method. Single bacterial colonies were inoculated in the TS broth at 37 °C with shaking for 48 h. Bacteria were collected by centrifugation at 10,000 rpm for 1 min and washed twice with sterile deionized water. Total genomic DNA was extracted and purified by the GENEray™ Bacteria Whole Genome DNA Extraction Kit GK1072 (Generay Biotech, Shanghai, China). The DNA purity and concentration was measured by a NanoDrop™ spectrophotometer. DNA fragmentation was conducted using an ultrasound machine. DNA fragments of around 500 bp size were separated and collected using Agarose Gel Electrophoresis. Finally, one DNA library was constructed using the Illumina TruSeq™ (Illumina, San Diego, CA, USA) DNA Sample Prep Kits for the draft genome sequencing. The same procedure was conducted to construct another DNA library for the complete genome sequencing by a different experimenter one year later.