Hello all,

I am currently working on the CNV calls for a infertile patients whole exome cohort.

So I want to make germline CNV calls on the latter.

I wanted to test CNVkit for this.

We have patients identified as positive controls and validated experimentally, possessing a WDR66 homodeletion on exons 20/22 to 22/22.

However with the following pipeline, from the segmentation stage I don't find a segment consistent with this target modification ...(hmm and hmm_germline method tested).

The intervals list comes from the Ensembl canonical transcripts list (delimited according to the exons size).

PIPELINE used:

cnvkit.py access ~/hs38DH.fa -x ~/sv_blacklist.bed -o ~/STEP1_ACCESS/acces-excludes.hg38.bed

cnvkit.py target ~/Interval_GRCH38_97_200921_STEP0.bed  --split -o ~/STEP2_TARGET_ANTITARGET/my_targets.bed
cnvkit.py antitarget ~/STEP2_TARGET_ANTITARGET/my_targets.bed -g ~/STEP1_ACCESS/acces-excludes.hg38.bed -o ~/STEP2_TARGET_ANTITARGET/my_antitargets.bed

test=$(find ~/BAM_FOLDER/ -name *.bam)
for bam in $test
do 
    name=${bam:0:-4}
    cnvkit.py coverage $bam -p 20 ~/STEP2_TARGET_ANTITARGET/my_targets.bed -o ~/STEP3_COVERAGE/$name.targetcoverage.cnn
    cnvkit.py coverage $bam -p 20 ~/STEP2_TARGET_ANTITARGET/my_antitargets.bed -o ~/STEP3_COVERAGE/$name.antitargetcoverage.cnn
done

cnvkit.py reference -o ~/STEP4_REFERENCE/FlatReference.cnn -f /data/septiera/REF_GENOME/hs38DH.fa -t ~/STEP2_TARGET_ANTITARGET/my_targets.bed -a ~/STEP2_TARGET_ANTITARGET/my_antitargets.bed

cnvkit.py fix ~/STEP3_COVERAGE/POSControlSample.targetcoverage.cnn ~/STEP3_COVERAGE/POSControlSample.antitargetcoverage.cnn ~/STEP4_REFERENCE/FlatReference.cnn -o ~/STEP5_FIX/POSControlSample.cnrhere

    cnvkit.py segment ~/STEP5_FIX/POSControlSample.cnr -m hmm -p 10  -o ~/STEP6_SEGMENT/POSControlSample_hmm_wdrop.cns

cnvkit.py segmetrics ~/STEP5_FIX/POSControlSample.cnr -s ~/STEP6_SEGMENT/POSControlSample_hmm_wdrop.cns --ci -o ~/STEP7_CALL/POSControlSample_hmm_wdrop_ci.cns

cnvkit.py call ~/STEP7_CALL/POSControlSample_hmm_wdrop_ci.cns --filter ci -y -o ~/STEP7_CALL/POSControlSample_call_ci.cns

POSControlSample.cnr result example:

chromosome  start   end gene    log2    depth   weight
chr12   121918592   121918695   -   -6.34236    3.82524 0.0001     #EXON1
chr12   121921286   121921683   -   4.49197 141.151 0.0001     #other gene
chr12   121923622   121923990   -   4.08991 124.815 0.0001     #EXON2
chr12   121931746   121931886   -   2.21378 107.421 0.0001     #EXON3
chr12   121934247   121934356   -   -1.77108    69.9817 0.0001
chr12   121942534   121942645   -   0.540772    138.306 0.0001
chr12   121942895   121942975   -   -0.904748   106.25  0.0001
chr12   121948984   121949061   -   3.90008 48.9091 0.0001
chr12   121951480   121951530   -   1.33628 69.5    0.0001
chr12   121954120   121954334   -   2.44978 76.4579 0.0001
chr12   121957074   121957268   -   3.89977 164.613 0.0001
chr12   121958272   121958522   -   3.93186 170.108 0.0001
chr12   121958943   121959094   -   2.87873 135.053 0.0001
chr12   121960585   121960758   -   0.838862    59.8035 0.0001
chr12   121961978   121962162   -   0.077442    30.2228 0.0001
chr12   121966955   121967069   -   0.574232    83.2719 0.0001
chr12   121968006   121968169   -   -0.284698   122.264 0.0001
chr12   121975244   121975334   -   -3.15683    27.9889 0.0001
chr12   121975542   121975685   -   1.88913 93.4965 0.0001
chr12   121976185   121999216   Antitarget  -0.857707   0.588164    0.896216
chr12   121999716   121999944   -   -23.0153    0   0.0001     #EXON20  
chr12   122001497   122001598   -   -6.13142    1.61386 0.0001   #EXON21
chr12   122003652   122003919   -   3.10711 61.7678 0.0001     #EXON22

POSControlSample_hmm_wdrop.cns result example:

chromosome  start   end gene    log2    depth   probes  weight
chr12   121917290   121942645   -   -4.14401    58.6302 10  0.001 #WDR66 SEGMENT 1 EXONS 1/22-6/22
chr12   121942895   122018922   -   -0.783351   0.707287    20  1.74422 #WDR66 SEGMENT 2 EXONS 7/22-22/22

POSControlSample_call_ci.cns result example:

chromosome  start   end gene    log2    cn  depth   probes  weight
chr12   121917290   121942645   -   -4.14401    0   58.6302 10  0.001  #EXONS 1/22-6/22 HOMODEL (error)
chr12   121942895   122216869   -   -0.48987    1   0.932991    117 5.27485   #EXONS 7/22-22/22 HETDEL (error)

Is my pipeline correct for this analysis type? How can I improve it?

Thanks in advance!!