Shotgun Metagenome Analysis

Metagenomics analysis is done to understand the dynamics of microbial communities from an environmental sample and it provides classification of species level taxonomy and the projection of metabolic pathway activities from microbial samples. Shotgun Metagenome Analysis generally includes sequencing of the complete metagenome samples followed by de novo assembly of multiple genomes from sequence reads of multiple species in an environmental sample. During assembly the paired end reads are compared to each other, and then overlapped reads are used to build longer contiguous sequences. The contiguous sequences are further analyzed for prediction of genes and functional annotations.

Workflow and deliverables

Bioinformatics workflow for Shotgun Metagenome Analysis:

a. Quality check of raw reads:

The raw reads will be subjected to quality filtration and adapter trimming. The primer sequences, poly(A) tails and reads produced from ribosomal DNA templates will be removed. The high quality data will be used for downstream analysis.

b. De novo Assembly

The denovo assembly of high quality reads will be carried out using assembler. The PE data will be assembled using various Kmer length, coverage cutoff, insert length, insert length standard deviation, expected coverage for scaffold assembly. The best assembly will be selected based on scaffold N50 and max scaffold length. Then final assembly will be evaluated based on scaffolds N50, assembly coverage (depth), assembly completeness and accuracy.

c. Gene prediction

Ab initio gene predictors are statistical models which are trained to find features of genes, such start and stop codons, CDS of the genes. The assembly contiguous sequences will be used as input in gene prediction program to predict the coding region in the given sample.

d. Taxonomic analysis

Taxonomic analysis of the predicted genes is carried out, with a program of sensitive taxonomic classification. It uses either the set of available complete genomes from NCBI RefSeq or the microbial subset of the NCBI BLAST non-redundant protein database “nr”, optionally also including fungi and microbial eukaryotes.

e. Functional Annotation

Functional annotation of the genes are performed using a standalone tool which provides COG, KEGG, Pfam, GO and SEED subsystem annotations to individual sequences constituting metagenomic datasets.

f. Comparative analysis

The comparative analysis will be done when we have more than one sample. The samples will be compared based on the abundance count and functionally annotated genes.

Deliverables

• Quality filtration of data
• Denovo assembly of high quality reads
• Gene prediction
• Taxonomic identification
• Abundance and identification of microbial community,
• Functional annotation
• COG, Pfam, KEGG, FIG and GO subsystem annotations
• KEGG pathway,
• Krona plot
• NCBI submission (SRA)
• Comparative analysis (if samples is more than one)
• Comprehensive report with publication standard methodology, graphs and tables.