title: Raw V3-V4 amplicon of sediment DNA isolated from riverbed sediment of Tulsi River
design: DNA extraction from the soil samples was done by DNeasy Power Soil Pro Kit ( QIAGEN 201324) and the extracted DNA was first seen on 0.8% agarose gel (Fig-1) and quantified by Nanodrop 1000 (Thermo Fisher Scientific, 3411 Silverside Road Bancroft Building, Suite 100, Wilmington, DE 19810 U.S.A). Then the V3-V4 region of the 16s rDNA of the extracted DNA was amplified by HIMEDIA Prima-96 (Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate, Thane (West) - 400604, Maharashtra, India.) using V3-V4primer (CCTAYGGGRBGCASCAG as forward primer and GGACTACNNGGGTATCTAAT as reverse primer). The amplicons were then visualized on 2% agarose gel (Fig-2) and a Pure gene Nexgen DNA ladder (PG210500DI-NV) was used as a marker. The DNA library was prepared by NEBNext Ultra II DNA Library Prep Kit (NEB #E7645S/L), the libraries were then quantified by Qubit 4.0 fluorometer (Thermofisher #Q33238) using DNA HS assay kit (Thermofisher #Q32851) according to manufacturer protocol. To identify the insert size, a query on Tapestation 4150 (Agilent) utilizing high-sensitive D1000 screentapes (Agilent # 5067- 5582) following the manufacturers protocol was done. sequencing was done on the Illumina (NOVASEQ6000) platform using NovaSeq 6000 S4 Reagent Kit v1.5. Raw data quality assessment was performed using FastQC v.0.11.9 and followed by report generation using Fastp v.0.23.4 [41]. The data was preprocessed using BBDuk v.38.86 (Parameters: qtrim=rl, trimq=18, tbo=f) as per the published recommendation by Mohsen et all  Primer fragments of matched sequences were removed with the cutadapt plugin  Sequences were then quality filtered, denoised, merged and the chimera removed, using the DADA2 plugin in QIIME2  SILVA database (Release 138) was downloaded and sequences flanking the forward and reverse primers were extracted followed by training the QIIME2 Nave Bayes feature classifier  The representative sequences were classified by taxon using the fitted classifier. QIIME2 was executed with standard parameters, with DADA2 quality settings with --p-trunc-len-f 284 --p-trunc-len-r 185. Amplicon Sequence Variants (ASVs) annotated as mitochondria or chloroplasts were removed by QIIME2 export options because these sequences are typically considered unwanted. Relative abundance was calculated based on the taxonomic classification. An iterative abundance of the taxa was plotted using a barplot concerning their samples. Further downstream analysis was performed on the biom file. Stacked bagplots based on relative abundance were plotted using the Microeco R packages. Alpha rarefaction curve and alpha diversity (parameters: - Group=SampleTypes, Measures= Chao1, Shannon, Simpson, ACE, Observed OTUs) were plotted using the Phyloseq R package. The heatmap was plotted based on percentage read abundance using Ampvis2 R packages. Krona chart was plotted using the psadd R package.  Predictive functional analysis of bacterial communities was done using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2)  Gene families for COG, EC, KO, and PFAM pathways were predicted and annotated.
library_name: S191123B