title: Metagenomics analysis for Larva gut of Tenebrio Molitor (Meal worm)
design: Protocol:DNA Extraction : (Optional)DNA extraction is done using the suitable method for the sample type from commercially available kits such as QIAGEN, ZYMO RESEARCH, ThermoFisherDnaextraction was done as per the manufacturer's recommendation.PCR Amplification of V3-V4 Region of 16s Gene:Composition of TAQ Master MIX:1) High-Fidelity DNA Polymerase 2) 0.5mM dNTPs3) 3.2mM MgCl24) PCR Enzyme BufferPrimer Details:16sF:-5 AGAGTTTGATGMTGGCTCAG316sR:-5 TTACCGCGGCMGCSGGCAC3Conditions Used:40ng of Extracted DNA is used for amplification along with 10pM of each primerInitial Denaturation:95 degree C25 Cycles of the following condition:Denaturation @ 95 degC for 15 sec, Annealing @ 60 degC for 15 sec, Elongation @ 72 degC for 2 minsFinal Extension at 72 degC for 10 minsand Hold at 4 degC.Overview of Sequencing Protocol:The ampliconsfrom each sample were purified with Ampurebeads to remove unused primers and an additional 8 cycles of PCR was performed using Illuminabarcoded adapters to prepare the sequencing libraries. Libraries were purified using Ampurebeads and quantitated using QubitdsDNAHigh Sensitivity assay kit. Sequencing was performed using IlluminaMiseqwith 2x300PE v3 sequencing kit.Bioinformatics protocol: Raw data QC is done using FASTQC and MULTIQC, followed by trimming of adapters and low quality reads by TRIMGALORE.The trimmed reads are further taken for processing which includes merging of paired end reads, chimeria removal and OUT abundance calculation and estimation correction this is achieved by QIIME / MOTHUR / KRAKEN / BRACKEN workflows. This workflow enables highly accurate investigations at genus level.The databases used are SILVA / GREENGENES / NCBIEach read is classified based on % coverage and identity. The 16S workflow will be useful in identifying pathogens in a mixed sample or understanding the composition of a microbial community.DNA QC :Extracted DNA from the samples were subjected to Nano Drop and GEL Check before being taken for PCR amplification:The Nano Drop readings  of 260/280 at an ~ value of 1.8 to 2 is used to determine the DNAs qualityPCR AmpliqonQC:The amplified 16s PCR Product is purified and subjected to GEL Check and NanodropQCThe Nano Drop readings  of 260/280 at an ~ value of 1.8 to 2 is used to determine the DNAs quality