Library Construction & Sequencing: Quality Control
The Northwest Genomics Center (NWGC) performs multiple QC steps on DNA and RNA samples prior to sequencing.
All samples are tested using the Quant-iT Broad Range dsDNA kit (Invitrogen) to measure DNA concentration or the Qubit RNA assay kit (Invitrogen) to measure RNA concentration.
Sample Sex Assay
For genotyping assays, all DNA samples are tested using an ABI TaqMan assay specific for X/Y chromosomal regions. A definitive sex genotype is generated for each sample. These data will be compared against the reported sex from the submitted sample manifest.
A custom OpenArray genotyping assay is performed on all exome and WGS samples to generate a 63 SNP molecular fingerprint. These data are used to confirm the gender reported on the manifest and to check concordance with the final sequencing data.
Fragment Analysis for RNA samples
All RNA samples are run using an Advanced Analytical Fragment Analyzer to assess the initial quality of each sample. An RQS score is generated for each sample and these data are compared against the reported RQS from the submitted sample manifest.
The investigator will receive an email and a report detailing any QC failures. Samples that fail QC are not eligible to proceed into the sequencing pipeline. QC issues will need to be resolved by the investigator before the project can proceed to sequencing.
Resolution of QC Failures
If a sample has failed due to a sex conflict or due to insufficient concentration, the investigator may submit a replacement sample by filling out a new manifest describing the replacement sample. Submission of replacement samples will delay the release of data for the project.
If a replacement sample is not available, a single sample may be excluded from the project. In cases where the failing sample is critical to the analysis (i.e., the proband in a trio), the entire family will be excluded.
OpenArray fingerprinting is performed using the Thermo Fisher QuantStudio 12k Flex rt-PCR with the AccuFill System. The fingerprint assay design consists of 64 high-frequency allele SNPs which offer a unique signature for each sample and the ability to confirm the genders of submitted samples to their manifest entries. Samples must pass the minimum call rate threshold of 90% to be further processed in the pipeline. These SNP data will be used to later assure concordance with sample sequencing data to ensure tracking integrity has been maintained.
Prior to sequencing, DNA samples are genotyped using the following method:
64 high frequency, exome-specific SNPs using custom-made OpenArray plates via QuantStudio 12K.