title: RNA-Seq of sediment: sample20
design: The extracted DNA was used as a template for polymerase chain reaction (PCR), whereby the V4 variable region of the 16S rRNA gene was amplified using the 515/806 primers, with barcode on the forward primer. The HotStarTaq Plus Master Mix Kit (Qiagen, USA) was used for 30 PCR cycles. Using unique dual indices, equal proportions of the samples were pooled together to multiplex the samples based on their DNA concentrations and molecular weight, after which the samples were purified using calibrated Ampure XP Beads. An Illumina DNA Library was then prepared using the resulting purified PCR product. A Miseq was used to perform sequencing according to the manufacturers instructions, followed by the processing of the sequence data through MR DNA ribosomal and functional gene analysis pipeline. The analysis pipeline comprised joining the sequences as well as removing sequences with less than 150 base pairs (bp) and/or ambiguous base calls. The obtained sequences were filtered to improve their quality by setting a maximum expected error threshold of 1.0, after which they were dereplicated. The resulting unique sequences were then denoised, whereby the sequences attributed to sequencing and/or PCR point errors were removed.
identifiers.namespace: SUB15199585
identifiers.id: T42_28