Step 1 : GE Preflight Checks (QC)

  • Updated

Tapestri’s GE Pipeline starts with running a quality check (QC) module, which checks the validity of the input data. This QC module ensures that the FASTQ files used are valid and have no issues that might cause GE Pipeline to fail. It also detects the chemistry and sets it as the run chemistry for the downstream analysis of the DNA Pipeline. If all files pass QC, GE Pipeline moves on to the next steps.

FASTQ files generated from the Illumina sequencer are checked for quality using the fastp module. The file format, as well as sequence quality, is verified before running the pipeline.

Failure of QC

Under certain conditions, the QC may fail, and in every event, the run status displays a suitable message. The following table summarizes the errors along with the run status and reason for the error.

Issue Run Status Reason
QC failed due to FASTQ file corruption QC Failed
  • FASTQ file is not a valid .gz archive.
A sample is oversequenced QC Failed - Oversequenced Sample


 

  • This check is only applicable if the panel contains more than 100 amplicons
  • The expected coverage for the FASTQ file containing the read pairs is more than 160x, where
    expected_coverage = (readpair_count * lane_count) / (expected_num_of_cells = 30000 * amplicon_count)
QC failed due to high percent of Ns at a position QC Warning
  • There are more than 20% Ns at a particular read position across all reads.
Barcode Version cannot be determined QC Warning
  • Fixed part counts for v1 and v2/v3 barcode versions are insufficient to reliably determine the version of the sample (v2 and v3 chemistry use the same barcode structure). 

R1/R2 read mismatch

 

 QC Failed
  • The R1 and R2 contain different number of reads.
Amplicon Overlap  QC Failed
  • All chromosomes in the panel are not available in the genome fasta file.
Protein panel issues QC Failed
  • Mandatory columns - Name, ID, Sequence - are missing
  • Sequence contains non-ATGC characters
  • Duplicate barcode sequence
  • Invalid column separator
  • non UTF-8 file encoding

Based on the reasons above, investigate the reason for the run failure or contact support@missionbio.com for additional help.

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