Amplicon Sequence Variant (ASV) processing pipeline

Tools: MultiQC DADA2 phyloseq

Load necessary packages

library(dada2) library(ggplot2) library(phyloseq)

Set filepath

path <- 'Path/to/files' list.files(path) # verify all of the files of interest are present.

Sort files into forward and reverse reads.

F_reads <- sort(list.files(path, pattern='_R1_001.fastq')) R_reads <- sort(list.files(path, pattern='_R2_001.fastq'))

Obtain sample names from the filenames

sample.names <- sapply(strsplit(F_reads, '_'), [, 1)

Set full path for read files

F_reads <- file.path(path, F_reads) R_reads <- file.path(path, R_reads)

Create directory for filtered and trimmed read files

filtered_path <- file.path(path, 'filtered')

Rename the newly filtered and trimmed files

F_filtered <- file.path(filtered_path, paste0(sample.names, '_F_filtered.fastq.gz')) R_filtered <- file.path(filtered_path, paste0(sample.names, '_R_filtered.fastq.gz'))

Filtering and Trimming

output <- filterAndTrim(F_reads, F_filtered, R_reads, R_filtered, truncLen=c(290,290), # truncLen should be set based on fastQC results maxN=0, maxEE=c(2,2), truncQ=2, rm.phix=TRUE, compress=TRUE, multithread=TRUE) head(output)

Estimating Error Rate

errorRate_F <- learnErrors(F_filtered, multithread=TRUE) errorRate_R <- learnErrors(R_filtered, multithread=TRUE)

Visualizing the error rates

plotErrors(errorRate_F, nominalQ=TRUE) plotErrors(errorRate_R, nominalQ=TRUE)

Dereplicate reads to improve computation

derep_F <- derepFastq(F_filtered, verbose=TRUE) derep_R <- derepFastq(R_filtered, verbose=TRUE) names(derep_F) <- sample.names names(derep_R) <- sample.names

Sample Inference

F_inference <- dada(derep_F, err=errorRate_F, multithread=TRUE) R_inference <- dada(derep_R, err=errorRate_R, multithread=TRUE)

Merging Reads (exact overlap)

merged <- mergePairs(F_inference, derep_F, R_inference, derep_F, verbose=TRUE)

Create Sequence Table

seq_table <- makeSequenceTable(merged) table(nchar(getSequences(seq_table))) # Provides count of sequence variants of various lengths