Single_copy_genes, ValueError: invalid contig Gene1

[ebhmayra]

Hello,

I have a new issue with DEVIATE, I'm trying to use it with --single_copy_genes normalization. I included 5 genes sequences in the --library file and then I call them for the normalization, but I got errors "ValueError: invalid contig Gene1". Could you help me to find out what is wrong, please?

This is my script:

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deviaTE --input_bam $data_folder/minimifoliaBT131_aligned.bam --families rnd_1_family_1_Unknown,rnd_1_family_334_LTR_Copia,rnd_1_family_54_LTR_Gypsy,rnd_3_family_201_Unknown,rnd_4_family_797_LTR_Gypsy,rnd_4_family_195_LTR_Gypsy,rnd_4_family_256_Unknown,rnd_4_family_342_LTR_Gypsy,rnd_4_family_694_LINE_RTE_BovB,rnd_5_family_1219_LINE_RTE_BovB,rnd_5_family_1298_LTR_Gypsy,rnd_5_family_1744_LTR_Gypsy,rnd_6_family_1113_Unknown,rnd_5_family_989_Unknown,rnd_6_family_3088_LTR_Gypsy,rnd_6_family_8663_LTR_Gypsy --single_copy_genes Gene1,Gene2,Gene3,Gene4,Gene5 --library $data_folder/impolita-families_mcclintok9_cdhit2_cat.fa

---

This is the error:

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**** Analysis Starting analysis of rnd_1_family_1_Unknown in /scratch/botany/mayra/diospyros/minimifoliaBT131_aligned.bam.fused.sort.bam..

No annotaions found for: rnd_1_family_1_Unknown Reference sequence contains ambiguous nucleotide: N Reference sequence contains ambiguous nucleotide: N Reference sequence contains ambiguous nucleotide: N Reference sequence contains ambiguous nucleotide: N Reference sequence contains ambiguous nucleotide: N Reference sequence contains ambiguous nucleotide: N Reference sequence contains ambiguous nucleotide: N Normalization: single copy genes

Traceback (most recent call last): File "/home/user/bricenohuayta/.conda/envs/deviaTE_env/bin/deviaTE_analyse", line 113, in scg_fac = sample.get_norm_fac_scg(genes=args.single_copy_genes) File "/home/user/bricenohuayta/.conda/envs/deviaTE_env/lib/python3.6/site-packages/deviaTE/deviaTE_pileup.py", line 162, in get_norm_fac_scg sum_cov = sum([len(pileupcolumn.pileups) for pileupcolumn in bamfile_op.pileup(contig=g, truncate=True)]) File "pysam/libcalignmentfile.pyx", line 1322, in pysam.libcalignmentfile.AlignmentFile.pileup File "pysam/libchtslib.pyx", line 687, in pysam.libchtslib.HTSFile.parse_region ValueError: invalid contig Gene1

**** Visualization

During startup - Warning message: Setting LC_CTYPE failed, using "C" Warning message: Input file does not exist

---

Best regards, Mayra

[W-L]

Hi thanks for getting in touch! Could you check if the names of the single copy genes you are using appear in the same way:

• in the alignment file. You can check with samtools view -H <file.bam>. All of them need to appear as a @SQ line in the bam header
• in the library fasta: check with grep '>' <library.fa>
• on the command line

The spelling etc. needs to be exactly the same between these three occurrences, otherwise it won't be matched up correctly. Since the error is coming from pysam, my guess is that there is something wrong or missing from the bam file. Did you include the same single copy genes when you aligned the reads?

Best wishes, Lukas

[ebhmayra]

Hi Lukas,

This was useful. Thank you for your help. Best, Mayra