PureCN Coverage.R error

Question

[cg_ref_database]

I'm using the PureCN package to calculate GC-normalized coverage, but I'm receiving the following error below (bottom of page).

After execution is halted, a compressed bed-type text file is produced that has intervals for each of the chromosomes:

chr1:925918-926037 2150 25 1 0.880952380952381
chr1:930126-930365 4548 36 1 0.898016997167139
chr1:931005-931124 3449 41 1 0.871875

Can someone help me troubleshoot what the problem is and how to fix the error?

INFO [2020-07-12 13:30:25] Loading PureCN 1.18.0...
INFO [2020-07-12 13:30:26] Processing 
WARN [2020-07-12 13:30:35] Found 398 overlapping intervals, starting at line 2422.
WARN [2020-07-12 14:52:25] tumor.coverage.file and interval.file do not align.
WARN [2020-07-12 14:52:25] Intervals in coverage and interval.file have conflicting on/off-target annotation.
Error in simpleLoess(y, x, w, span, degree = degree, parametric = parametric,  :
  invalid 'x'
Calls: .gcNormalize ... correctCoverageBias -> .correctCoverageBiasLoess -> loess -> simpleLoess
Execution halted

[markus.riester]

Looks like there are still some issues with the interval files. Can you share the command line and output of both IntervalFile.R and Coverage.R?

[cg_ref_database]

The interval file command was:

Rscript IntervalFile.R \
--infile Exome_v1_hg38_Probes_Standard_REMOVE_five.bed \
--fasta GRCh38.d1.vd1.fa \
--outfile ref_check_output/baits_hg38_intervals_hg38probes_grch38.d1.vdl.txt \
--offtarget --genome hg38 \
--export ref_check_output/baits_optimized_hg38_Exome_v1_hg38_Probes_Standard_REMOVE_five_AND_GRCh38.d1.vd1.bed \
--mappability GCA_000001405.15_GRCh38_no_alt_analysis_set_100.bw

The output was:

INFO [2020-07-13 01:49:17] Loading GCA_000001405.15_GRCh38_no_alt_analysis_set_100.bw...
INFO [2020-07-13 01:50:18] Loading PureCN 1.18.0...
INFO [2020-07-13 01:50:18] Processing Exome_v1_hg38_Probes_Standard_REMOVE_five.bed...
WARN [2020-07-13 01:50:19] Found 27938 overlapping intervals, starting at line 38.
WARN [2020-07-13 01:50:22] Target intervals were not sorted.
INFO [2020-07-13 01:50:30] Splitting 16804 large targets to an average width of 400.
INFO [2020-07-13 01:50:59] Tiling off-target regions to an average width of 200000.
INFO [2020-07-13 01:50:59] Removing following contigs from off-target regions: chr4_GL000008v2_random,chr14_GL000009v2_random,chr14_GL000225v1_random,chr15_KI270727v1_random,chr16_KI270728v1_random,chr17_KI270729v1_random,chrUn_KI270442v1,chrUn_KI270743v1
WARN [2020-07-13 01:52:03] 268 intervals without mappability score (268 on-target).
INFO [2020-07-13 01:52:03] Removing 5079 intervals with low mappability score (<0.50).
WARN [2020-07-13 01:52:04] No reptiming scores provided.
INFO [2020-07-13 01:52:04] Calculating GC-content...
Warning message:
In .Seqinfo.mergexy(x, y) :
  Each of the 2 combined objects has sequence levels not in the other:
  - in 'x': CMV
  - in 'y': chr11_KI270721v1_random, chr14_GL000194v1_random, chr14_KI270722v1_random, chr14_KI270723v1_random, chr14_KI270724v1_random, chr14_KI270725v1_random, chr14_KI270726v1_random, chr17_GL000205v2_random, chr17_KI270730v1_random, chr1_KI270706v1_random, chr1_KI270707v1_random, chr1_KI270708v1_random, chr1_KI270709v1_random, chr1_KI270710v1_random, chr1_KI270711v1_random, chr1_KI270712v1_random, chr1_KI270713v1_random, chr1_KI270714v1_random, chr22_KI270731v1_random, chr22_KI270732v1_random, chr22_KI270733v1_random, chr22_KI270734v1_random, chr22_KI270735v1_random, chr22_KI270736v1_random, chr22_KI270737v1_random, chr22_KI270738v1_random, chr22_KI270739v1_random, chr2_KI270715v1_random, chr2_KI270716v1_random, chr3_GL000221v1_random, chr5_GL000208v1_random, chr9_KI270717v1_random, chr9_KI270718v1_random, chr9_KI270719v1_random, chr9_KI270720v1_random, chrEBV, chrM, chrUn_GL000195v1, chrUn_GL00021 [... truncated]
Loading required package: TxDb.Hsapiens.UCSC.hg38.knownGene
Loading required package: GenomicFeatures
Loading required package: AnnotationDbi
Loading required package: org.Hs.eg.db

WARN [2020-07-13 01:54:37] Attempted adding gene symbols to intervals. Heuristics have been used to pick symbols for overlapping genes.
Warning messages:
1: In .Seqinfo.mergexy(x, y) :
  Each of the 2 combined objects has sequence levels not in the other:
  - in 'x': chr1_GL383518v1_alt, chr1_GL383519v1_alt, chr1_GL383520v2_alt, chr1_KI270759v1_alt, chr1_KI270760v1_alt, chr1_KI270761v1_alt, chr1_KI270762v1_alt, chr1_KI270763v1_alt, chr1_KI270764v1_alt, chr1_KI270765v1_alt, chr1_KI270766v1_alt, chr1_KI270892v1_alt, chr1_KN196472v1_fix, chr1_KN196473v1_fix, chr1_KN196474v1_fix, chr1_KN538360v1_fix, chr1_KN538361v1_fix, chr1_KQ031383v1_fix, chr1_KQ458382v1_alt, chr1_KQ458383v1_alt, chr1_KQ458384v1_alt, chr1_KQ983255v1_alt, chr1_KV880763v1_alt, chr1_KZ208904v1_alt, chr1_KZ208905v1_alt, chr1_KZ208906v1_fix, chr1_KZ559100v1_fix, chr2_GL383521v1_alt, chr2_GL383522v1_alt, chr2_GL582966v2_alt, chr2_KI270767v1_alt, chr2_KI270768v1_alt, chr2_KI270769v1_alt, chr2_KI270770v1_alt, chr2_KI270771v1_alt, chr2_KI270772v1_alt, chr2_KI270773v1_alt, chr2_KI270774v1_alt, chr2_KI270775v1_alt, chr2_KI270776v1_alt, chr2_KI270893v1_alt, chr2_KI270894v1_alt, chr2_KN538362v1_fix, chr2_KN538363v1_ [... truncated]

[cg_ref_database]

(continued)

2: In .Seqinfo.mergexy(x, y) :
  Each of the 2 combined objects has sequence levels not in the other:
  - in 'x': chr1_GL383518v1_alt, chr1_GL383519v1_alt, chr1_GL383520v2_alt, chr1_KI270759v1_alt, chr1_KI270760v1_alt, chr1_KI270761v1_alt, chr1_KI270762v1_alt, chr1_KI270763v1_alt, chr1_KI270764v1_alt, chr1_KI270765v1_alt, chr1_KI270766v1_alt, chr1_KI270892v1_alt, chr1_KN196472v1_fix, chr1_KN196473v1_fix, chr1_KN196474v1_fix, chr1_KN538360v1_fix, chr1_KN538361v1_fix, chr1_KQ031383v1_fix, chr1_KQ458382v1_alt, chr1_KQ458383v1_alt, chr1_KQ458384v1_alt, chr1_KQ983255v1_alt, chr1_KV880763v1_alt, chr1_KZ208904v1_alt, chr1_KZ208905v1_alt, chr1_KZ208906v1_fix, chr1_KZ559100v1_fix, chr2_GL383521v1_alt, chr2_GL383522v1_alt, chr2_GL582966v2_alt, chr2_KI270767v1_alt, chr2_KI270768v1_alt, chr2_KI270769v1_alt, chr2_KI270770v1_alt, chr2_KI270771v1_alt, chr2_KI270772v1_alt, chr2_KI270773v1_alt, chr2_KI270774v1_alt, chr2_KI270775v1_alt, chr2_KI270776v1_alt, chr2_KI270893v1_alt, chr2_KI270894v1_alt, chr2_KN538362v1_fix, chr2_KN538363v1_ [... truncated]
3: In .Seqinfo.mergexy(x, y) :
  Each of the 2 combined objects has sequence levels not in the other:
  - in 'x': CMV, chrEBV
  - in 'y': chr1_GL383518v1_alt, chr1_GL383519v1_alt, chr1_GL383520v2_alt, chr1_KI270759v1_alt, chr1_KI270760v1_alt, chr1_KI270761v1_alt, chr1_KI270762v1_alt, chr1_KI270763v1_alt, chr1_KI270764v1_alt, chr1_KI270765v1_alt, chr1_KI270766v1_alt, chr1_KI270892v1_alt, chr1_KN196472v1_fix, chr1_KN196473v1_fix, chr1_KN196474v1_fix, chr1_KN538360v1_fix, chr1_KN538361v1_fix, chr1_KQ031383v1_fix, chr1_KQ458382v1_alt, chr1_KQ458383v1_alt, chr1_KQ458384v1_alt, chr1_KQ983255v1_alt, chr1_KV880763v1_alt, chr1_KZ208904v1_alt, chr1_KZ208905v1_alt, chr1_KZ208906v1_fix, chr1_KZ559100v1_fix, chr2_GL383521v1_alt, chr2_GL383522v1_alt, chr2_GL582966v2_alt, chr2_KI270767v1_alt, chr2_KI270768v1_alt, chr2_KI270769v1_alt, chr2_KI270770v1_alt, chr2_KI270771v1_alt, chr2_KI270772v1_alt, chr2_KI270773v1_alt, chr2_KI270774v1_alt, chr2_KI270775v1_alt, chr2_KI270776v1_alt, chr2_KI270893v1_alt, chr2_KI270894v1_alt, chr2_KN538362 [... truncated]
4: In .Seqinfo.mergexy(x, y) :
  Each of the 2 combined objects has sequence levels not in the other:
  - in 'x': CMV, chrEBV
  - in 'y': chr1_GL383518v1_alt, chr1_GL383519v1_alt, chr1_GL383520v2_alt, chr1_KI270759v1_alt, chr1_KI270760v1_alt, chr1_KI270761v1_alt, chr1_KI270762v1_alt, chr1_KI270763v1_alt, chr1_KI270764v1_alt, chr1_KI270765v1_alt, chr1_KI270766v1_alt, chr1_KI270892v1_alt, chr1_KN196472v1_fix, chr1_KN196473v1_fix, chr1_KN196474v1_fix, chr1_KN538360v1_fix, chr1_KN538361v1_fix, chr1_KQ031383v1_fix, chr1_KQ458382v1_alt, chr1_KQ458383v1_alt, chr1_KQ458384v1_alt, chr1_KQ983255v1_alt, chr1_KV880763v1_alt, chr1_KZ208904v1_alt, chr1_KZ208905v1_alt, chr1_KZ208906v1_fix, chr1_KZ559100v1_fix, chr2_GL383521v1_alt, chr2_GL383522v1_alt, chr2_GL582966v2_alt, chr2_KI270767v1_alt, chr2_KI270768v1_alt, chr2_KI270769v1_alt, chr2_KI270770v1_alt, chr2_KI270771v1_alt, chr2_KI270772v1_alt, chr2_KI270773v1_alt, chr2_KI270774v1_alt, chr2_KI270775v1_alt, chr2_KI270776v1_alt, chr2_KI270893v1_alt, chr2_KI270894v1_alt, chr2_KN538362 [... truncated]

The coverage command was:

Rscript Coverage.R --outdir coverage_output/ \
--bam 01.GRCh38.d1.vd1.SortSam.MarkDuplicates.recalibrated.bam \
--intervals ref_check_output/Exome_v1_hg38_Probes_Standard_REMOVE_five_AND_GRCh38.d1.vd1.txt

The output was:

INFO [2020-07-13 02:27:43] Loading PureCN 1.18.0...
INFO [2020-07-13 02:27:43] Processing 01.GRCh38.d1.vd1.SortSam.MarkDuplicates.recalibrated_coverage.txt.gz...
WARN [2020-07-13 02:27:53] Found 398 overlapping intervals, starting at line 2422.
WARN [2020-07-13 03:49:35] tumor.coverage.file and interval.file do not align.
WARN [2020-07-13 03:49:35] Intervals in coverage and interval.file have conflicting on/off-target annotation.
Error in simpleLoess(y, x, w, span, degree = degree, parametric = parametric,  :
  invalid 'x'
Calls: .gcNormalize ... correctCoverageBias -> .correctCoverageBiasLoess -> loess -> simpleLoess
Execution halted

[markus.riester]

Weird. That’s a new one. Can you share the input BED file? Via github issue or email.

But removing the overlaps as mentioned in the warning should fix it. It should do it automatically, but I guess some intervals are repeated multiple times and merged only once.

[cg_ref_database]

Yes, definitely, can I get the email account to send the BED file to? Also, just to be clear, is that the probes BED file?