Genotyping a sample

It is very easy to use STRipy. To genotype a sample, please select the following on the home screen:

1. Select from drop-down list either a Disease or Locus to target, or specify coordinates and motif manually into the Custom field.
2. Choose the aligned and indexed BAM or CRAM file of the Sample you want to genotype
3. If the selected locus is in one of the sex chromosomes, then select the selected sample's Sex (or let STRipy to detect this automatically, but always double-check the result)
4. Select the name of the Genome assembly which was used to align the selected BAM or CRAM file (or let STRipy to detect this automatically)
5. Choose the type of Analysis which can be either Standard or Extended:
   • Standard: fast analysis (often between 5-15 seconds) which is recommended to use at the first try. Maximum detectable repeat size is limited to the fragment length.
   • Extended: slower analysis (usually between 20 seconds to few minutes) which can be used when you want to expand the genotyping ability past the fragment length. This could be good to try when STRipy notifies of a potentially long allele or when you suspect an allele that is at least ~few hundred bp long. NB! Analysis of long STR alleles from short-read sequencing data is very complicated and often results either a under- or overestimated allele. Use this option carefully.
6. Pick a Server where the genotyping takes place. Unless you have set up your own server, then Cloud is the only activated option.

Viewing results

The following colour scheme is used which is based on data acquired from the literature:

- determined length of an allele is in the normal range;
- determined length of an allele is in the intermediate range;
- determined length of an allele is in the pathogenic range;
- determined length of an allele is none of the above ranges and therefore considered to be as in the "unknown" range.

Pink dot in the "Genotype" section is used to mark an allele that is a population outlier (Z-score ≥3.718). Population data is acquired by genotyping 1000 Genomes Project cohort.

STRipy's pipeline version is a non-graphical command-line version which is merged from STRipy's Server and Client. The pipeline version can be integrated into pipelines and can genotype multiple loci at the same time. Analysis report can be saved as HTML file for easy viewing of results. Click here to see a demo report!
If you have any requests or suggestions regarding the pipeline version and/or the outputted HTML report, then don't hesitate to contact the author.

Please see the installation instructions and configuration parameters on gitlab.com/andreassh/stripy-pipeline. We implemented a flag system to spot the potentially interesting loci.

API address: https://api.stripy.org

Annotate an ExpansionHunter's VCF file

It is easy to annotate VCF files with disease names, inheritance and the range into which the repeat size falls using the command line. This can be done with 'curl' by specifying the VCF file which will be sent through STRipy's online annotation program. The same VCF file is returned where the loci that match with the ones in STRipy's database is annotated.

Example request and response:
POST /annotateVCF/file HTTP/1.1
Host: api.stripy.org

##fileformat=VCFv4.1
##INFO=<ID=DISID,Number=1,Type=String,Description="Disease ID as specified in the STRipy database">
##INFO=<ID=DISNAME,Number=1,Type=String,Description="Disease name as specified in the STRipy database">
##INFO=<ID=DISINHER,Number=1,Type=String,Description="Disease inheritance mode as specified in the STRipy database">
##INFO=<ID=DISRANGE,Number=1,Type=String,Description="Range for both allele repeats for the disease as specified in the STRipy database">
...header truncated...
#CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	HG04225
chr9	27573528	.	C	<STR8>,<STR29>	.	PASS	END=27573546;REF=3;RL=18;RU=GGCCCC;VARID=C9ORF72;REPID=C9ORF72;DISID=FTDALS1;DISNAME=Amyotrophic lateral sclerosis and/or frontotemporal dementia;DISINHER=AD;DISRANGE=Normal/Pathogenic	GT:SO:REPCN:REPCI:ADSP:ADFL:ADIR:LC	1/2:SPANNING/INREPEAT:8/29:8-8/26-36:17/0:32/57:0/4:43.378378
...other loci...

Example command using curl and my_sample.vcf file; output will be saved as my_sample.annotated.vcf:
curl -F 'file=@my_sample.vcf' https://api.stripy.org/annotateVCF > my_sample.annotated.vcf

Create a variant catalogue for ExpansionHunter

Create a variant catalogue from the API input. For example, this allows to generate it from the command line via using wget or curl:
wget -O HTT_only.json https://api.stripy.org/catalogue/hg38/on/off/HTT
or
curl https://api.stripy.org/catalogue/hg38/on/off/CSTB,EIF4A3,PRNP,VWA1 > vntr_catalogue.json.
The genomes can be either "hg19", "hg38", or "hs1". To include "chr" in chromosome names, set the "use_chr_prefix" value to "on"; otherwise, use "off". To include off-target regions, set the "use_offtargets" value to "on"; if not, use "off". Specify a list of loci, separated by commas, to be included in the catalogue.

GET /catalogue/{genome}/{use_chr_prefix}/{use_offtargets}/{loci_ids} HTTP/1.1
Host: api.stripy.org

Example request and response:
GET /catalogue/hg38/on/off/HTT,DMPK,FXN HTTP/1.1

Retrieve information about a locus

Retrieves information about a locus (for example, HTT) in the database, including coordinates, motif and linked diseases. Notes: "IntermediateRange" is null if no intermediate range is specified; "Motif" and "MotifPlusStrand" is an array for RFC1 locus; NIPA1 locus has additional "GenomeExceptions" key for motif sequence on hg19 genome.

GET /locus/{locus_id} HTTP/1.1
Host: api.stripy.org

Example request and response:
GET /locus/HTT HTTP/1.1

Compare repeat length against the database

Compares the number of repeats (for example, 40 or 20/22) against the literature (under "Literature" key) and population data (under "Population" key). "Zscore" is calculated comparing the repeats to our population data, and IsOutlier returns true if Z-score is at least 3.718 (1 in 1000). If no standard deviation can be calculated then Zscore and IsOutlier turns to null. In case multiple diseases are linked to one locus, results for each of them will be returned (see the second example). One or two repeat lengths/alleles can be used.

GET /compare/{locus_id}/{repeats} HTTP/1.1
Host: api.stripy.org

Example (1) request and response:
GET /compare/HTT/40 HTTP/1.1

Using two repeats/alleles
Instead of one numerical value, both alleles can be specified which are separated by slash, for example: 5/6.

GET /compare/{locus_id}/{repeats1}/{repeats2} HTTP/1.1
Host: api.stripy.org

Example (2) request and response:
GET /compare/COMP/5/6 HTTP/1.1