Homework 7 Report

This is the repo for the homeworks of the BI-2024 Practicum Course

Supplementary materials for project:

Metagenomic analysis of the ancient oral microbiome from the oral cavity of fossil human remains

by Dmitriy Matach and Kirill Petrikov

Content

16S

MAG

Terminal commands

 

16S

Additional files for 16S rRNA amplicone metagenome sequence:

• teeth.Rmd - R Markdown-script for DADA2-analisys
• reads_quality_control.png - DADA2 quality control results for each sample
• reads_quality_control_aggregate.png - aggregated DADA2 quality control results
• asv_table.csv - final ASV-tabel from DADA2-analisys
• tax_table.csv - ASV taxonomy assignment for MicrobiomeAnalyst
• metadata.csv - samples metadata for MicrobiomeAnalyst
• norm_libsizes_0.png - MicrobiomeAnalyst library size plot for input samples

Additional R-script for visualization of ASVs corresponding to "red complex" bacteria

library(dplyr)
library(reshape2)

barplotdata <- ps@otu_table[, c(150, 135, 243, 129, 177, 327, 397)]
bpdata <- melt(barplotdata)

bpdata <- bpdata %>%
  mutate(affliction = case_when(
    Var1 == 'SRR986773.fastq' ~ "Peridontitis and the red complex",
    Var1 == 'SRR986774.fastq' ~ "Peridontitis and the red complex",
    Var1 == 'SRR986778.fastq' ~ "Peridontitis",
    Var1 == 'SRR986779.fastq' ~ "Peridontitis",
    Var1 == 'SRR986782.fastq' ~ "Peridontitis",
    .default = 'Unafflicted')
    )

p <- ggplot(bpdata, aes(x = Var1, y = Var2)) + 
  geom_point(aes(size = value, col = affliction)) + 
  scale_x_discrete(guide = guide_axis(angle = 90)) + 
  xlab('read') + 
  ylab('ASV')

p

 

MAG

Additional files for metagenome assembled genomes analisys:

• sankey_plot.html - Sankey diagram by Pavian for MAGs taxonomy assignment results
• ref_unique_genes.gff - Features selected after intersection as unique for Tannerella forsythia modern reference genome
• KofamKOALA_result.txt - results of KofamKOALA search on selected proteins unique for reference genome

 

Terminal commands used for MAG analisys

Create index of referense genome, MAGs contigs alignment

bwa index NC_016610.fasta && \
bwa mem -t 8 NC_016610.fasta G12_assembly.fna | \
samtools view -b --threads 8 | \
samtools sort --threads 8 > alignment.sorted.bam

Get alignment statistics

samtools flagstat alignment.sorted.bam

Create bed-file from bam-file

bedtools bamtobed -i alignment.sorted.bam > alignment.bed

Intersect referense genome and MAGs contigs, keeping only unique for referense genome

bedtools intersect -v -a NC_016610.gff3 -b alignment.bed > intersect.gff

Select only CDS, filter out «hypothetical» and «pseudo» proteins, keep only transposases and all but transposases

awk '{FS="\t";OFS="\t"} $3 ~ "CDS"' intersect.gff | grep -v 'product=hypothetical' | grep -v 'pseudo=true' | grep 'transposase' > transp.gff

awk '{FS="\t";OFS="\t"} $3 ~ "CDS"' intersect.gff | grep -v 'product=hypothetical' | grep -v 'pseudo=true' | grep -v 'transposase' > cds_inters.gff

Select proteins IDs for further NCBI Entrez batch download and KofamKOALA-analisys

cut -f 9  cds_inters.gff | cut -f 1 -d ";" | cut -f 2 -d "=" | grep -o -P "WP_\d+.\d" > prot_idxs.txt