Troubleshooting
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      1. AION guide
      2. AION - Instructions for Use
      3. Troubleshooting

      Troubleshooting

      General recommendations

      Additionally to the VCF format checklist in VCF format , these are things to consider to guarantee best results:

      • Does your VCF file contain variants with enough quality?

        • All variants in the VCF file which do not fulfil the following quality criteria will be filtered out by AION to avoid evaluating false positive variants. This means that even if your file has the required format and fields, it may contain low quality data, and wrong or no results would be produced.

        • Small variants:

          • Depth (DP) > 10x

          • Variant allele frequency (VAF) > 20%

        • CNV / SV variants:

          • FILTER = PASS

        • AION CNV analysis does not hard filter on other quality metrics due the high variability of the scales of these metrics between different variant callers. However, AION CNV analysis considers as high quality and ranks higher variant fulfilling the criteria outlined above (VCF format | CNV / SV variants VCFs ).

      • What variant callers can be used to generate VCF files?

        • In principle all variant callers can be used to generate the VCF for small variants, but some tools do not normalise variant calls by default (e.g. contain multi-allelic variants that have not been standardised following best practices). This can result in variant annotation problems: please revise that variant calls have been normalised before submitting the files.

        • For CNVs and SVs, AION has been validated on Canvas, Manta, Dragen CNV and Dragen SV. Other variant callers may be supported if they adhere to the same standards.

      • Shall I apply any variant filtering before submitting the files?

        • Best results are guaranteed when low-quality and artifactual variants (generated during the sequencing process) have been filtered out from VCF files.

      Error codes

      In case there is an error, the user is informed through the following messages:


      Error code 

      Category

      Explanation

      What to do 

      E103

      General

      Failed on decompressing input file. Only gzipped and bgzipped vcf files are supported

      Use another tool to compress the VCF file or contact support if needed

      E104

      General

      Some of the provided gene symbols or HGNC IDs are invalid

      Validate the input and contact support if needed

      E140

      VCF integrity

      Provided samples do not have unique sample IDs. There are more than one files with the same sample ID

      Merge the VCFs with repeated IDs and submit again

      E141

      VCF integrity

      Required fields check failed for input file! There are some missing fields in the VCF that are required

      See required fields above and contact support if needed

      E142

      VCF integrity

      Import check failed for input file

      Contact support

      E143

      VCF integrity

      VCF file could not be checked for mandatory columns

      Contact support

      E144

      VCF integrity

      VCF file is missing the some columns

      See required fields above and contact support if needed

      E145

      VCF integrity

      VCF file truncated

      Validate the input and contact support if needed

      E146

      VCF integrity

      GVCF file detected.

      GVCF is currently not a supported input format, please convert to VCF. Contact support if needed

      E147

      VCF integrity

      Structural variant file submitted to SNV pipeline or the other way around.

      See information for how to submit a case Creating a case , contact support if needed.

      E150

      VCF Processing

      VCF consistency checks failed due to missing definitions in header

      See required fields above and contact support if needed

      E151

      VCF Processing

      No variants in the selected region. Update your in silico panel filters if they were applied

      If you added in silico panel filters, resubmit the case selecting other genes, otherwise the content of you VCF may not overlap with AION’s supported region. Contact support if needed.

      E162

      Multisample VCF

      Found more than one sample in input VCF! Currently, AION only supports singletons or trios.

      Split your VCF into individual samples and submit again. Contact support if needed.

      E170

      Reference genome mismatch

      A mismatch was found between the reference genome provided and the input VCF files

      Verify the provided reference genome is correct

      E301

      Sample matching

      The sample ID in the small and structural variants VCFs doesn’t match for the same samples.

      Verify the sample IDs in the VCF files
       

      Additional resources: