ExomeDepth error in getBamCounts when adding a fasta reference

Question

[Francesco]

Whenever I try to add a reference fasta file for computing the GC content in the GetBamCounts function:

my.countsV6 <- getBamCounts(bed.frame =AgilentV6,
                            bam.files = BAMFiles,
                            include.chr = TRUE,
                            referenceFasta = "data/hg19.fa"
                          )

I get an error like this:

Reference fasta file provided so ExomeDepth will compute the GC content in each window Error in value[[3L]](cond) : record 38397 (chr11:134201957-134202041) failed file: data/hg19.fa

Everything works fine without the referenceFasta. I tried both with the UCSC and Ensembl build but I get the same error on a different chromosome.

R version 3.6.3 (2020-02-29)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 18363)

Matrix products: default

locale:
[1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252    LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C                           LC_TIME=English_United States.1252    

attached base packages:
[1] parallel  stats4    stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] GenomicRanges_1.38.0 GenomeInfoDb_1.22.1  IRanges_2.20.2       S4Vectors_0.24.4     BiocGenerics_0.32.0 
[6] ExomeDepth_1.1.15    readr_1.3.1

Answer 1

[Francesco]

Solved! It's very likely it was an overflow problem that R encounters with files bigger than 2GBs (as the hg19 fasta file) I manually computed the GC content of all the exonic regions with bedtools:

$ bedtools nuc -fi hg19.fa -bed S07604514_hs_hg19/S07604514_Covered_edit.bed | awk '{$4=""; print $0}' > AgilentV6_exons_GC_count.txt

Than I manually added the GC contents column to the my.countsV6 dataframe

[sonsunjirachote]

Hello

I just got the same error as your. Could you clarify after calculated GC content?

How did you manually add GC contents column to your dataframe?

Thanks in advance

JK