Estimate sizes of repeats in a especific Gene

Question

[Rafael]

Amateur problem here:

We know that it is possible to use the ExpansionHunter tool to estimate sizes of such repeats by performing a targeted search through a BAM/CRAM file for reads that span, flank, and are fully contained in each repeat. Here is the reference link: https://github.com/Illumina/ExpansionHunter

Such repeat regions can expand to a size much larger than the read length and thereby cause a disease. Fragile X Syndrome, ALS, and Huntington's Disease are well known examples.

But I have encountered some difficulties dealing with ExpansionHunter.

I ask: is it possible to search for repetitions using other software, such as IGV (Integrative Genomics Viewer) for example or any other equivalent software?

[Rafael]

Tell me if I'm in the way. I have the CRAM file and the respective CRAI (index).

So I just ran the SAM like this, clipping my area of interest:

$ samtools view -b NG1PSZ7BE9.mm2.sortdup.bqsr.cram "chrX:147912050-147912110" > result.bam

Then I indexed the .bam file:

$ samtools index result.bam

My reference file is this:

http://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/chrX.fa.gz

My variants file looks like this:

[    
{
      "LocusId": "FMR1",
      "LocusStructure": "(CGG)*",
      "ReferenceRegion": "chrX:147912050-147912110",
      "VariantType": "Repeat"    
} 
]

When trying to run ExpansionHunter...:

$ ./Hunter/bin/ExpansionHunter --reads result.bam --reference chrX.fa --variant-catalog variant_catalog.json --output-prefix output

I get the following error:

2023-05-17T17:23:01,[Starting ExpansionHunter v5.0.0] 2023-05-17T17:23:01,[Analyzing sample result] 2023-05-17T17:23:01,[Initializing reference chrX.fa] 2023-05-17T17:23:01,[Loading variant catalog from disk variant_catalog.json] 2023-05-17T17:23:01,[Running sample analysis in seeking mode] 2023-05-17T17:23:01,[Analyzing FMR1] 2023-05-17T17:23:01,[Could not recover the mate of NG1PSZ7BE9_2023_04_13_192298_ProPhase_BP_FP200009316L1C044R06203103192/1] 2023-05-17T17:23:01,[Writing output to disk]

[Pierre Lindenbaum]

you didn't read my comment above Estimate sizes of repeats in a especific Gene . At this point I'm leaving the thread. Sorry.

[WouterDeCoster]

But I have encountered some difficulties dealing with ExpansionHunter.

If you could elaborate on that...

[Rafael]

I'm going to start from the beginning to make sure I don't miss any steps.

I already have ExpansionHunter properly installed and running.

I have the patient's .CRAM file with his 30x WGS that is approximately 60 gigabytes in size.

I think that first I need to cut the area of interest referring to the desired expansion in FMR1, correct?

First of all, probably using samtools, I need to clip the specific region of FMR1 where Fragile-X repeats occur: chrX:147912050-147912110

Something like that?

samtools view input.cram "chrX:147912050-147912110" > output.cram

[Pierre Lindenbaum]

the command above produces a SAM file without header and is missing a reference genome to decode the CRAM.

you want samtools view -T /path/to/ref.fa -O CRAM -o output.cram input.cram "chrX:147912050-147912110"

[Pierre Lindenbaum]

and if you want to use expansion hunter in a defined region, just create a variant-catalog containing the region and use expansion with --analysis-mode seeking https://github.com/Illumina/ExpansionHunter/blob/master/docs/03_Usage.md

[Rafael]

Cool, I am going to try.

Doubt: Is it mandatory to extract the desired region using samtools or can I run ExpansionHunter directly with the original .CRAM file?

[Pierre Lindenbaum]

I already answered in my comment above.

[Rafael]

Perfect, do you suggest any reference FASTA files for the human genome that you tend to use frequently?