inst/extdata/intro.md

In Manuelaio/uncoverappLib: Interactive graphical application for clinical assessment of sequence coverage at the base-pair level

An interactive graphical web application for clinical assessment of sequence coverage at base-pair level

This is a web application for clinical assessment of sequence coverage. unCOVERApp allows:

• to display interactive plots showing sequence gene coverage down to base-pair resolution and functional/ clinical annotations of sequence positions within coverage gaps (Coverage Analysis page).

• to calculate the maximum credible population allele frequency (AF) to be applied as AF filtering threshold tailored to the model of the disease-of-interest instead of a general AF cut-off (e.g. 1 % or 0.1 %) (Calculate AF by allele frequency app page).

• to calculate the 95 % probability of the binomial distribution to observe at least N variant-supporting reads (N is the number of successes) based on a user-defined allele fraction that is expected for the variant (which is the probability of success). Especially useful to obtain the range of variant-supporting reads that is most likely to occur at a given a depth of coverage (DoC)(which is the number of trials) for somatic variants with low allele fraction (Binomial distributionpage).

Documentation

All unCOVERApp functionalities are based on the availability of a bed file containing tab-separated specification of genomic coordinates (chromosome, start position, end position), coverage value and reference: alternate allele counts read for each position. In the first page Preprocessing, users prepare the bed file providing input files containing a list of genes and a list of bam files, respectively:

• a text file, with .txt extension, containing HGNC official gene name(s) one per row and to be uploaded to Load input file box. An example file is included in extdata of uncoverappLib packages. Users can use also a target bed with 4 columns: chromosome, start, end and gene/amplicon

Below is an example of genes list.

DNAJC8
GNB1
PEX10
RPL22

• a text file, with .list extension, containing absolute paths to BAM files (one per row) and to be uploaded to Load bam file(s) list box. In the output file, sample 1,2,3.., correspond to the sample in the bam file bam.list file listed in row 1,2,3, …. Below is an example of bam.list in a Unix-like OS including macOS.

/home/user/bam/sample1.bam
/home/user/bam/sample2.bam
/home/user/bam/smaple3.bam

While all inputs are loading, a progress bar appears during processing phase. unCOVERApp input file generation fails if incorrect gene names are specified. An unrecognized gene name(s) table is displayed if such a case occurs.

Below is a snippet of bed file output of Preprocessing unCOVERApp.

chr15   89859516        89859516        68      A:68
chr15   89859517        89859517        70      T:70
chr15   89859518        89859518        73      A:2;G:71
chr15   89859519        89859519        73      A:73
chr15   89859520        89859520        74      C:74
chr15   89859521        89859521        75      C:1;T:74

The preprocessing time depends on the size of the BAM file(s) and on the number of genes to investigate. In general, if many (e.g. > 50) genes are to be analyzed, we would recommend using buildInput function and run it in R console before launching the app.

Alternatively, other tools do a similar job and can be used to generate the unCOVERApp input file ( for instance: bedtools, samtools, gatk).

In this case, users can load the file directly on Coverage Analysis page in Select input file box.

Once bed file is ready, users can move to Coverage Analysis page for own analysis and push load prepared input file button.

To assess sequence coverage the following input parameters must be specified in the sidebar of the Coverage Analysis section:

• Reference Genome : reference genome (hg19 or hg38)

• Gene name and push Apply button: HGNC official gene name

• coverage threshold : required coverage threshold

• Sample : sample(s) to analyze according to help text indications on the App page

• Transcript number : transcript number as in first column of Exon genomic coordinate positions from UCSC output App table.

• exon number and push Make exon : to zoom in a specific exon

Other input sections, as Chromosome, Transcript ID, START genomic position, END genomic position and Region coordinate, are dynamically filled.

unCOVERApp generates the following outputs :

• unfiltered BED file inbed file and the corresponding filtered dataset in Low coverage positions

• UCSC gene coordinates in UCSC gene table

• exon coordinates and transcript number in UCSC exon table

• sequence gene coverage plot in Gene coverage. The plot displays chromosome ideogram, genomic location and gene annotations from Ensembl and transcripts annotation from UCSC. Related table shows the number of uncovered position in each exon given a user-defined transcript number

• plot of a specific exon, selected by sidebar exon number , in Exon coverage. Related table shows the number of Low coverage positions annotate in ClinVar and with a high impact annotation.

• dbNSFP annotation of low coverage positions can be found in Annotation on low-coverage positions . By clicking on the download button, users can save the table as spreadsheet format with certain cells colored according to pre-specified thresholds for AF, CADD, MAP-CAP, SIFT, Polyphen2, ClinVar, OMIM ID, HGVSp and HGVSc, ...).

• In Calculate maximum credible allele frequency page, users can set allele frequency cut-offs based on specific assumptions about the genetic architecture of the disease. Users may click on the download button and save the resulting table as spreadsheet format.

• The Binomial distribution page returns the 95 % binomial probability distribution of the variant supporting reads on the input genomic position (START genomic position and END genomic position). Users should define the expected allele fraction (the expected fraction of variant reads, probability of success) and Variant reads (the minimum number of variant reads required by the user to support variant calling, number of successes).

Manuelaio/uncoverappLib documentation built on Feb. 16, 2023, 12:52 a.m.