Truseq nano dna sample preparation protocol

DNA quality and quantity were determined using a 2100 Bioanalyzer and the Invitrogen PicoGreen assay, respectively. Library preparation was performed according to the Illumina TruSeq Nano DNA Sample preparation protocol. The samples were sheared on a Covaris S220 (Covaris, Woburn, MA, USA) to ∼450 bp, following the manufacturer’s recommendation, and each uniquely tagged with one of Illumina’s TruSeq LT DNA barcodes. Sequencing was performed on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) resulting in an average of 56.18 million (49.29–77.79 M) 250 bp paired-end reads per sample. Sequence data were deposited in the Sequence Read Archive and are available under the BioProject PRJNA886972.

Illumina TruSeq Nano DNA sample preparation protocol was used for sequencing library preparation. Genomic sequencing of Thauera samples was performed on a MiSeq (Illumina, USA); genomic sequencing of UPWRP_1 and UPWRP_2 samples was performed on a HiSeq 2500 (Illumina, USA). Adaptors were removed, and reads were quality trimmed with a minimum Phred score of 20 and a minimum length of 30 bp from both ends using the software BBDuk tools (BBMap–Bushnell B. http://sourceforge.net/projects/bbmap).

Total DNA was extracted with the FastDNA^® Spin Kit for Soil (MP Biomedicals, Solon, USA) according to the manufacturer’s protocol. DNA concentration was assessed by Nanodrop 2000 (Thermo Scientific, Wilmington, USA) and DNA quality was determined by 1% agarose gel electrophoresis.
For 16S rRNA sequencing, primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) were used to amplify the V3-V4 hypervariable regions of the bacterial 16S rRNA gene using PCR on a thermocycler (GeneAmp 9700, ABI, USA). The final PCR products were extracted from a 2% agarose gel, purified by the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluorTM -ST (Promega, USA). When purification and quantification were finished, a compound of amplicons were merged into equimolar concentrations and paired-end sequenced (2 × 300) on Illumina MiSeq PE300 (Illumina, San Diego, USA).
For metagenomic shotgun sequencing, total DNA prepared from saliva samples were sheared on Covaris S220 (Covaris, Woburn, MA, USA) to an average size of 400 bp. Library preparation was performed according to Illumina’s TruSeq Nano DNA Sample Preparation protocol, followed by sequencing on Illumina HiSeq 2500 (Illumina, San Diego, USA).
The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP336507).