Nebnext ultra dna library prep kit

The Listeria spp. isolates obtained from sand samples were cultured in the BHI agar plate at 37 °C for 18 h. After incubation, the bacterial cells from one agar plate were collected via scraping followed by genomic DNA extraction using a Wizard^® Genomic DNA Purification Kit (Promega; Madison, WI, USA). The extracted DNA was quantified using a Qubit 2.0 Fluorometer. The DNA library was constructed using the NEBNext^® UltraTM DNA Library Prep Kit (NEB; 240 County Road, Ipswich, UK) with Illumina technology. The whole-genome shotgun sequencing of these isolates was performed on the Illumina Hiseq PE150 platform. The raw data were filtered to obtain clean data, and then high-quality paired reads were assembled using SOAP denovo (v 2.04). Genome sequencing, raw data filtration, and clean data assembly were performed using the instruments from Novogene Co., Ltd. (Beijing, China). For species-level identification, fastANI (v 1.33) was used to calculate the average nucleotide identity (ANI) between the genomes. Prokka (v 1.14.6) was used for genome annotation tasks.

The two T. meditationis specimens used in this study were collected in Xian, Shaanxi, China and stored in liquid nitrogen until mRNA extraction. Using the NEBNext UltraTM DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA), two cDNA libraries (T1 and T2) were constructed from two T. meditationis individuals following the manufacturer’s instructions and sequenced on an Illumina HiSeq 2500 high-throughput sequencing platform (Illumina, San Diego, CA, USA). Raw sequencing data were uploaded to the NCBI database (project number: PRJNA912169, SRA number: SRR22734937).
After quality filtering, reads from each library were assembled in Trinity 2.4.0 (http://trinityrnaseq.github.io/ (accessed on 21 July 2020) with default parameters [53 (link)]. The transdecoder function was used to extract transcripts with open reading frames (ORF) [54 (link)]. Using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 28 July 2021), we compared the transcripts assembled from the two libraries and only retained those that exist in both libraries (i.e., having an blastx match in the other library with an E-value < 1 × 10⁻⁵⁰) [55 (link)].

Microbial DNA was extracted following the protocol included in the QIAamp DNA stool kit (Qiagen). The quality and quantity of the extracted DNA were measured using NanoDrop (Thermo Scientific) and Qubit (Thermo Fisher Scientific, Singapore). The DNA fragment size was evaluated by agarose gel electrophoresis. Then, the short-insert DNA libraries were generated using a NEBNext^® UltraTM DNA Library Prep Kit (NEB, USA), and we executed 2 × 150bp paired-end sequencing via an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, United States).

The V3-V4 region of the 16S rRNA gene was amplified using the primers 341F (5-GTGCCAGCMGCCGCGGTAA-3) and 805R (5-ACTACHVGGGTATCTAATCC-3). Polymerase chain reactions (PCRs) were performed using Phusion^® High-Fidelity PCR Master Mix (New England Biolabs) and the amplicons generated were confirmed by gel electrophoresis, purified with Qiagen Gel Extraction Kit (Qiagen, Germany) and quantified using a Qubit^®3.0 Fluorometer (Invitrogen, Thermo Fisher Scientific). The final library including barcodes and adaptors was generated with the NEBNext^® UltraTM DNA Library Prep Kit. The amplicons were then sequenced using Illumina sequencing (NovaSeq 6000) at Novogene (Beijing, China). The sequence data obtained were deposited in the Sequence Read Archive (SRA), under accession number PRJNA933568.

Additional metagenomes were generated from novel samples obtained from the following environmental sites: deep-sea sediment from Juan de Fuca Ridge in the Pacific Ocean off the coast of Canada; fumaroles and hot springs in the Azores; the Tatta Pani Hot Spring and Khewra Salt Mine, Pakistan; and four hot springs in Guangdong, China. More information on the sampling sites, including geographic coordinates and environmental parameters, is provided in the Supplementary Text.
Metagenomes were generated as previously described [59 (link)]. Briefly, genomic DNA was isolated from the samples using commercial kits with minor modifications. Quality and quantity of extracted DNA were determined with Nanodrop and Qubit spectrophotometers (Thermo Fisher Scientific Inc., Waltham, MA, USA). Metagenomic shotgun libraries of sheared DNA were prepared using the NEBNext^® UltraTM DNA Library Prep Kit (New England BioLabs, Frankfurt am Main, Germany). Libraries were sequenced on an Illumina NexSeq550 instrument (Illumina, San Diego, CA, USA).

Genomic DNA of C. culicis and P. polymyxa was extracted using the SDS method. Then, the DNA was measured using agarose gel electrophoresis and assessed using a Qubit®2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA). Purified genomic DNA was used to construct a sequencing library with the NEB Next® Ultra^TM DNA Library Prep Kit (NEB, Beverly, MA, USA). The genomes of C. culicis and P. polymyxa were sequenced on the Nanopore PromethION platform and Illumina NovaSeq PE150 (Beijing Novogene Bioinformatics Technology Co., Ltd, Beijing, China). After trimming low-quality reads using fastq, clean reads were assembled using SPAdes 3.13.1^{59 (link),81 (link)}. Bioinformatics analysis focused on KEGG Orthology^{82 (link)}. Genome overviews were created by Circos to reveal the annotations^{83 (link)}.

The total DNA was extracted from the insects' whole body tissue using the Tissue DNA Kit (Omega Georgia, Connecticut, USA) according to the manufacturer's instructions. The DNA content was quantified using a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Massachusetts, USA). A measure of 0.2 μg DNA was used as the input material for DNA library preparation. The sequencing library was prepared using the NEBNext UltraTM DNA Library Prep Kit (New England Biolabs, New York, USA) for Illumina according to the manufacturer's recommendations, and index codes were added. DNA fragments were then end-polished, A-tailed and ligated with the full-length adapter for Illumina sequencing, followed by further polymerase chain reaction (PCR) amplification. After PCR, the products were purified using the AMPure XP system (Beverly, Los Angeles, USA). The quality of the libraries was then assessed using the Agilent 5400 system (Agilent, Palo Alto, USA) and quantified by quantitative PCR (1.5 nm). The qualified libraries were pooled and sequenced on Illumina platforms with the PE150 strategy at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) according to the effective library concentration and the required amount of data.