Output

The directories and output files listed below can be found inside the the zip file. All paths are relative to the top-level results directory.

FastQC

FastQC (ver. 0.11.9) gives general quality metrics about your reads. It provides information about the quality score distribution across your reads and per base sequence content (%T/A/G/C). You get information about adapter contamination and other overrepresented sequences.

For further reading and documentation see the FastQC help.

The FastQC plots displayed in the MultiQC report shows untrimmed reads. They may contain adapter sequences and potentially regions with low quality.

Output directory: <RESULTS>/fastqc/

<SAMPLE>_fastqc.html

FastQC report, containing quality metrics for your untrimmed raw fastq files

Cutadapt

The pipeline uses Cutadapt (ver. 3.2) for removal of adapters, primers, poly-A tails and other types of unwanted sequences from high-throughput sequencing reads. If the option checkbox that allows for the possibility to skip the trimming step was marked (in case the samples to be processed have already had their primer sequences removed), these outputs will not be given.

Output directory: <RESULTS>/cutadapt/

<SAMPLE>.trimmed.cutadapt.log

Log file of the cutadapt process, with statistics regarding the primers trimmed.

cutadapt_summary.tsv

Statistics summary file for the whole run.

DADA2

DADA2 (ver. 1.20.0) is a fast and accurate algorithm for the inference of ASVs from amplicon data with single-nucleotide resolution.

The database that DADA2 will use to match the inferred ASVs against it and taxonomically classify them will be the Silva 138 database, due to its extended use in the field of metagenomics. The release 138 of the Silva database allows for the identification of a total 2,225,272 organisms, 1,983,534 of which are from the Bacteria kingdom. In Silva’s browser users can consult the contents of the database.

Output directory: <RESULTS>/dada2/

QC/

Internal quality control of the assignment, with several statistics and reports.

ASV_seqs.fasta

Fasta file of all the different species found in the samples with their OTU ID.

ASV_table.tsv

Table file with the number of different ASVs found in each sample.

ASV_tax.tsv

Table file with the maximum possible assigned taxonomy (Genus) for each ASV found and its fasta sequence.

ASV_tax_species.tsv

Table file with the maximum possible assigned taxonomy (Species) for each ASV found and its fasta sequence.

DADA2_stats.tsv

Table file statistics on taxonomy assignment.

DADA2_table.tsv and DADA2_table.rds

Table file containing the same information as the other tables, but in the specific format used for further analysis using R Studio and DADA2 package.

ref_taxonomy.txt

Information and citation for the database used (Silva 138).

QIIME2

QIIME2 (ver. 2021.2.0) is a next-generation microbiome bioinformatics platform. In this pipeline, several of its algorithms are used to further analyze ASV data obtained from the previous steps.

Output directory: <RESULTS>/qiime2/

abundance_tables/
abs-abund-table-X.tsv

Tab-separated absolute abundance table at taxa level X, ranging from 2 to 7 (Kingdom,Phylum, Class, Order, Family, Genus, Species).

count_table_filter_stats.tsv

Tab-separated table with information on how much counts were filtered for each sample.

feature-table.biom

Abundance table in biom format (general-use format for representing counts of observations in biological samples) for importing into downstream analysis tools.

feature-table.tsv

Tab-separated abundance table for each ASV and each sample.

rel_abundance_tables/: Absolute abundance tables produced by the previous steps contain count data, but the compositional nature of 16S rRNA amplicon sequencing requires sequencing depth normalization. This step computes relative abundance tables for various taxonomic levels and detailed tables for all ASVs with taxonomic classification, sequence and relative abundance for each sample. Typically used for in depth investigation of taxa abundances.
rel-abund-table-X.tsv

Tab-separated absolute abundance table at taxa level X, ranging from 2 to 7 (Kingdom,Phylum, Class, Order, Family, Genus, Species).

rel-table-ASV.tsv

Tab-separated relative abundance table for all ASVs.

rel-table-ASV_with-DADA2-tax.tsv

Tab-separated table for all ASVs with DADA2 taxonomic classification, sequence and relative abundance.

representative_sequences/
rep-seq.fasta

Fasta file with ASV sequences found in the samples.

descriptive_stats.tsv

Length, mean, etc. of ASV sequences.

seven_number_summary.tsv

Length of ASV sequences in different quintiles.

barplot/
index.html

Interactive barplot for taxa relative abundance per sample at the distinct taxonomic levels. Can be viewed in a web browser to aid exploration of taxa discovered in samples. Allows for sorting using associated metadata.

alpha-rarefaction/
index.html

Interactive plot of alpha rarefaction curves (one per metadata category) for taxa abundance per sample with distinct alpha diversity indices. Can be viewed in a web browser to determine if the richness of the samples has been fully observed.

phylogenetic_tree/
tree.nwk

Phylogenetic tree in newick format.

rooted-tree.qza

Phylogenetic tree in QIIME2 format.

diversity/
alpha_diversity/: Alpha diversity measures the species diversity within samples. It is calculated using various methods and pairwise comparisons of groups of samples are performed.
evenness_vector/index.html:

Pielou’s Evenness.

faith_pd_vector/index.html:

Faith’s Phylogenetic Diversity.

observed_otus_vector/index.html:

Observed OTUs.

shannon_vector/index.html:

Shannon’s diversity index.

beta_diversity/: Beta diversity measures the species community differences between samples. Distances are calculated using various methods and pairwise comparisons of groups of samples are performed.

<METHOD>_pcoa_results-PCoA/index.html: Interactive PCoA plot. Can be viewed in a web browser to assess and represent dissimilarity between samples. Method will be any of the following distance metrics methods: Binary Jaccard distance, Bray-Curtis distance, unweighted UniFrac distance, weighted UniFrac distance.

Krona plots

We use KronaTools (ver. 2.8.1) to create a very informative visualization of the absolute abundances of species for each sample, in HTML format to facilitate exploration.

Output directory: <RESULTS>/Krona_plots/

<SAMPLE>.plot.html

HTML report with the interactive krona plot for each sample of the run.

Sample krona plot:

Krona plot sample.

Sample report

Aggregated sample report in HTML format.

Output directory: <RESULTS>/Samples_report/

<SAMPLE>.html

Report with information about all steps per sample. It provides information about the number of sequences, sequences trimmed, assigned taxonomy and ASVs found.

Sample taxonomy assignment:

Taxonomy plot sample.

Analysis report

Aggregated report of all the samples, containing alpha and beta-diversity measures and a clustered heatmap.

Output directory: <RESULTS>/Run_report/

run.html

Sample clustered heatmap:

Heatmap plot sample.

MultiQC

MultiQC (ver. 1.10.1) is a visualization tool that generates a single HTML report summarizing all QC information for all the samples in your project.

The pipeline has special steps which allow the software versions used to be reported in the MultiQC output for future traceability.

Output directory: <RESULTS>/multiqc/

general.report.html

MultiQC report - a standalone HTML file that can be viewed in a web browser.

Pipeline information

The pipeline also provides a table listing software used and their respective versions.

Output directory: <RESULTS>/pipeline_info/

software_versions.tsv