Tapestri DNA 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 DNA 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, DNA Pipeline moves on to thenext steps.
For demultiplexing runs it also checks the validity of the sample variants file and the demultiplexing protein panel to prevent failures at a later stage.
FASTQ files generated from the Illumina sequencer are checked for quality using thefastpmodule. 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
Oversequenced Sample
v2 and v3 check - The expected coverage for the FASTQ file containing the reads is more than 320x, where expected_coverage = (read_count * tube_count * lane_count) / (expected_num_of_cells = 20000 * amplicon_count)
v3.4 check - 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 Failed (v2)
QC Warning (v3)
There are more than 20% Ns at a particular read position across all reads.
Barcode Version cannot be determined (Only v2/v3)
QC Failed
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).
Panel zip file is not correct
QC Failed
The panel file does not have the correct structure. See more details about the zip folder structure.
R1/R2 read mismatch (Only v3)
QC Failed
The R1 and R2 contain different number of reads.
Amplicon Overlap (Only v3)
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