2AS_mapping

Introduction

2AS_mapping performs the assembly with reference (i.e. mapping) of nucleotide sequences.

uml diagram

Run Analysis 2AS_mapping

Once the analysis 2AS_mapping has been selected from the run analyses interface, the user will be able to select which bioinformatic tool to use. The available tools are:

bowtie2 - Fast and sensitive read alignment
ivar - computational package for viral amplicon-based sequencing

Note: In case the software "snippy" is chosen, samples will need to have an associated 4AN_genes output files from "Prokka".

To select the mandatory reference genome, use the "Select reference" button in the input selection wizard. This will open a pop-up table, listing 2 different kind of sequences, both usable as reference:

reference fasta files;
consensus sequence or de novo assembly of a sample available in Cohesive.

With the tool "Ivar" it's possible to select multiple references (please consult the "Multiple references" section).

The input selection UI delivers an advanced input selection mode, to allow selection of all types of supported input files at once.

Accepted inputs can be from:

A link to Check analysis will be created after launching the requested analysis. The system will notify the user after a succesful analysis launch and once execution has ended.

Multiple references

If 2AS_mapping is performed with Ivar, it will be possible to select more than one reference, as shown in the image below. A guide to the multiple reference selection system is available at the corresponding section of the run analysis Wiki.

Output directory

Please refer to Cohesive's specific Wiki page for information on file download.

The output directory is available at the link in the download page or at the link presente in the analysis' summary card, and will have the following structure: results > YEAR > ID > 2AS_mapping > DSXXXXXXXX-DTXXXXXX_bowtie2. The last directory's suffix will be replaced with the name of the chosen tool. At that path there will be 2 directories:

meta: ("metadata") contains log and configuration files.
result: contains the analysis' output files.

The tables below list files produced by 2AS_mapping's available tools.

Bowtie2

File	Description	Location
DSXXXXXXXX-DTXXXXXX_ID_bowtie_REFID.fasta	consensus file	result directory
DSXXXXXXXX-DTXXXXXX_ID_bowtie_REFID.sorted.bam	bam (Binary Alignment Map) format alignment file	result directory
DSXXXXXXXX-DTXXXXXX_ID_bowtie_REFID.sorted.bam.bai	bai (bam file's index) file	result directory
DSXXXXXXXX-DTXXXXXX_ID_bowtie_REFID.var.flt.vcf	vcf (variant calling format) file with identified varaints	result directory
DSXXXXXXXX-DTXXXXXX_ID_bowtie_REFID_coverage_plot.png	coverage distribution graph	result directory

Ivar

Note: Ivar's execution consists of "Snippy", "Samtools" and "Ivar" tools execution.

File	Description	Location
DSXXXXXXXX-DTXXXXXX_ID_ivar_REFID.fasta	consensus sequence from Ivar	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.aligned.fa	reference with `-` in positions with `sequencing depth = 0`and `depth`'s `N` between 0 and the minimum number of reads considered for site coverage (no variants in this file)	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.bam	bam (Binary Alignment Map) format alignment file	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.bam.bai	bai (bam file's index) file	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.bed	bed (Browser Extensible Data) file	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.consensus.fa	reference genome with representation of all variants	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.consensus.subs.fa	reference genome with representation of substitution variants	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.txt	snippy run summary	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.tab	variant table in tsv format	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.csv	variant table in csv format	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.filt.vcf	variants filtered by Freebayes	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.raw.vcf	variants not filtered by Freebayes	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.subs.vcf	table of substitution variants in vcf format	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.gff	variants in GFF3 format	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.html	html version of the .tab table of variants	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.vcf	Snippy's output file with identified variants in vcf format	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.vcf.gz	snippy's vcf output file (archive)	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID.vcf.gz.csi	bcftools index of vcf.gz file	result directory
DSXXXXXXXX-DTXXXXXX_ID_vdsnippy_REFID_coverage_plot.png	coverage distribution graph	result directory

For further information about Snippy's output files, file formats and contents, please refer to Snippy's official manual.

Data visualization

Alignment of reads on the reference genome can be visualized with specific softwares (i.e. Tablet, BioEdit e uGene), which are able to read bam e bam.bai files: