Metagenomics (MiSeq-Illumina) Hydrothermal pretreated sugarcane bagasse
The 16S rRNA gene V3 and V4 variable region PCR  primers 341F-785R  with barcode on the forward primer  were used in a 28 cycle PCR  (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, after which a final elongation step at 72°C for 5 minutes was performed.  After amplification, PCR products are checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples are pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads. Then the pooled and purified PCR product is used to prepare illumina DNA library. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturers guidelines. The sequencing was done in Miseq - Illumina, 2x300 pb cycles. Sequence data were processed using MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA).  In summary, sequences were joined, depleted of barcodes then sequences <150bp removed, sequences with ambiguous base calls removed.  Sequences were denoised, OTUs generated and chimeras removed.  Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity).  Final OTUs were taxonomically classified using BLASTn against a curated database derived fromRDPII and NCBI (www.ncbi.nlm.nih.gov,  http://rdp.cme.msu.edu).
SUB2676385
https://submit.ncbi.nlm.nih.gov/subs/biosample/SUB2676352/overview