FOMC Micorbiome Pipeline Software Version and Commands (2024-12-01 V1.44)

DADA2 read denoising, merging and chimeral filtering:
    R version: 3.6.1 (2019-07-05)
    DADA2 version: 1.12.1
    DADA2: qualify profiles: plotQualityProfile
    Read subsampling: seqtk Version 1.3-r116-dirty
        seqtk sample -s100 R1|R2 5000
    Pairwise trimming and denoising test: L1 and L2 range from 10 to 50
        filterAndTrim(fnFs, filtFs, fnRs, filtRs, truncLen=c(L1,L2),trimLeft=c(19,19),maxN=0, rm.phix=TRUE,compress=TRUE, multithread=10)
        learnErrors(filtFs, multithread=10,errorEstimationFunction = loessErrfun)
        derepFastq(filtFs, verbose=TRUE)    
        dada(derepFs, err=errF, multithread=10)    
        mergePairs(dadaFs, derepFs, dadaRs, derepRs, verbose=TRUE)
        removeBimeraDenovo(seqtab, method="consensus", multithread=10, verbose=TRUE)
    Final trimming: best L1 and L2 from above
        filterAndTrim(fnFs, filtFs, fnRs, filtRs, truncLen=c(L1,L2),trimLeft=c(19,19),maxN=0, rm.phix=TRUE,compress=TRUE, multithread=10)
        learnErrors(filtFs, multithread=10,errorEstimationFunction = loessErrfun)
        derepFastq(filtFs, verbose=TRUE)
        dada(derepFs, err=errF, multithread=10)
        mergePairs(dadaFs, derepFs, dadaRs, derepRs, verbose=TRUE)
        removeBimeraDenovo(seqtab, method="consensus", multithread=10, verbose=TRUE)

Taxonomy Assignment:
    Nucleotide-Nucleotide BLAST 2.7.1+
    blastn -db DB -max_target_seqs 1000 -outfmt '6 qseqid sseqid qlen slen pident length mismatch gapopen qstart qend sstart send evalue bitscore' -query QUERY -out OUTFILE

Species-level chimera removal and novel specie OTU calling:
    USEARCH version v8.1.1861_i86linux32
        usearch -cluster_otus reads_sorted.fasta -otus OUTFILE -relabel OTU -otu_radius_pct 2 -uparseout UPARSEOUT -sizeout -log LOGOUT
    Nucleotide-Nucleotide BLAST 2.7.1+        
        blastn -db DB -max_target_seqs 1000 -outfmt '6 qseqid sseqid qlen slen pident length mismatch gapopen qstart qend sstart send evalue bitscore' -query QUERY -out OUTFILE

QIIME2 analyses:
    biom, version 2.1.5
        biom convert -i INFILE -o BIOMFILE --to-hdf5
    QIIME2 version qiime2-2020.11

Phyloseq analyses:
    phyloseq verson 1.28.0
        Import OTU
            otu=otu_table(as.matrix(otu_table),taxa_are_rows=TRUE)
            tax=tax_table(as.matrix(tax_table))
            sampledata=sample_data(meta_table)
            physeq=phyloseq(otu,tax,sampledata)
        aggregate at different taxonomy level
            psglom<-tax_glom(physeq,taxrank=level)
    microbiome version 1.6.0
        read count table transformation
            microbiome::transform(psglom, "compositional")
            microbiome::transform(psglom, "clr")
            microbiome::transform(psglom, "compositional")
        alpha diversity plots
            plot_richness(physeq,x="Group",measures=c("Observed","Shannon","Simpson"), title="Species Richness",color="Group") + geom_boxplot() + theme(plot.title = element_text(hjust = 0.5)) + xlab("Group")
        beta diversity plots
            ordinate(physeq,"NMDS","bray")
            ordinate(physeq,"PCoA","bray")
            ordinate(physeq,"NMDS","euclidean"")
            ordinate(physeq,"PCoA","euclidean")      
    microViz version 0.12.7
        taxonomy barcharts
            comp_barplot(phyloseq, tax_level = level, n_taxa = ntaxa, facet_by = compgroup)
       

Differential Abundance Analyses:
    ALDEx2 version 1.34.0
    microbiomeMarker version 1.8.0 ("run_aldex" command used to generate ALDEx2 result)
    LEfSe version 1.0.0-dev-e3cabe9
    ANCOMBC version 2.0.2