Assay Glossary
A list of available Clinical and Research Use Only (RUO) Assays and their descriptions available via our Project Initiation Form. Each assay involves the creation and analysis of whole genome libraries.
Whole Genome and Transcriptome Sequencing (WGTS)
This assay includes whole genome and whole transcriptome sequencing of a single tumour sample plus whole genome sequencing of a matched normal sample. Details on these assays are described in the relevant sections that follow:
- Minimum DNA input requirements as measured by fluorometric quantitation:
- 230 ng (genomic DNA from buffy coat)
- 80 ng (genomic DNA from fresh frozen)
- 130 ng (genomic DNA from FFPE)
- Minimum RNA input requirements as measured by fluorometric quantitation
- 80 ng (RNA from fresh frozen)
- 275 ng (RNA from FFPE)
- The is a 45 day turnaround time for clinical cases
- A clinical assay with geneticist-approved report is available in 80X/30X or 40X/30X whole genome coverage options depending on tumour cellularity (>65% required for 40X/30X)
- The clinical assay is also available for T/N pair WGS without associated transcriptome
- The clinical assay pricing includes extraction, the RUO pricing does not
Whole Genome Sequencing (WGS)
The KAPA Hyper Prep Kit (Roche) is used to create whole genome libraries. Initial library prep quality is assessed via visualization of size distribution on a Fragment Analyzer or TapeStation, and quantitated using a Qubit fluorometer. Library quality is determined using shallow MiSeq Sequencing to asses mapping to genomic regions. Libraries are then sequenced using 2X150 paired reads on an Illumina NovaSeq X Plus system to the requested target depth. Analysis proceeds through a validated pipeline after sequencing. FASTQ files called from the sequencing instrument are aligned using BWAmem.
Quality control measures are performed on both the raw and aligned reads, which are evaluated by internal staff to ensure they meet minimum metrics. The aligned file is pre-processed, marking duplicated and normalizing the alignment, prior to variant calling. The processed BAM file is used to call somatic copy number variants, purity and ploidy, loss of heterozygosity (LOH), structural variants, somatic SNVs (single nucleotide variants) and indels, and tumour mutation burden.
- RUO assay (data only) vailable in the following coverage options:
- 0.2X (shallow WGS for ichorCNA)
- 30X
- 40X
- 80X
- Minimum DNA input requirements as measured by fluorometric quantitation:
- 230 ng (genomic DNA from buffy coat)
- 80 ng (genomic DNA from fresh frozen)
- 130 ng (genomic DNA from FFPE)
- There is a 45 day turnaround time for clinical cases
- The clinical assay with a geneticist-approved report is available in 80X/30X or 40X/30X coverage options depending on tumour cellularity (>65% required for 40X/30X)
- The clinical assay pricing includes extraction: RUO pricing does not
Whole Transcriptome Sequencing (WTS)
Dual DNA/RNA extraction is performed by using the Qiagen Allprep DNA/RNA FFPE kit and quantitated using a Qubit fluorometer. The Illumina TruSeq Stranded Total RNA Library Prep Gold kit is used to create whole transcriptome libraries. During the library preparation, total RNA is depleted of ribosomal RNA (including mitochondrial ribosomal RNA), first and second strand cDNA is synthesized and tailed, adapters are litigated, and then the template is amplified using PCR.
Library quality is validated using shallow MiSeq sequencing to assess mapping to ribosomal sequencing and transcriptomic regions. Libraries are sequenced using 2×100 or 2×151 paired reads on an Illumina NovaSeq X Plus system to a target depth of 100M PE reads (clusters) per sample. Analysis proceeds through a validated pipeline after sequencing. FASTQ files called from the sequencing instruments are aligned using STAR (Spliced Transcripts Aligned to a Reference). Quality control measures are performed on both the raw and aligned reads, which are evaluated by internal staff to ensure they meet minimum metrics. The BAM (Binary Alignment Mapping) file is used to call fusion genes and evaluate percentile and gene expression outliers, as well as report immune infiltration.
- RUO assay (data only) available at 80M PE reads (clusters) coverage
- Minimum DNA input requirements as measured by fluorometric quantitation
- 80 ng (genomic DNA from fresh frozen)
- 275 ng (genomic DNA from FFPE)
Plasma Whole Genome Sequencing
The KAPA Hyper Prep Kit (Roche) is used to create whole genome libraries. Initial library prep quality is assessed via visualization of size distribution on a Fragment Analyzer or TapeStation, and quantitated using a Qubit Fluorometer. Library quality is determined using shallow MiSeq sequencing to assess mapping to genomic regions. Libraries are then sequenced using 2×150 paired reads on an Illumina NovaSeq X Plus system to the requested target depth. Anlaysis proceeds through a validated pipeline after sequencing.
- Clinical reporting
- The clinical assay with geneticist-approved report is available at 30X coverage; and as a follow-up to clinical WGTS or WGS assays
- Requires that Whole Genome sequencing data is available from the corresponding tumour with matched normal for reporting of Minimal Residual Disease (MRD)
- RUO FASTQ only
- FASTQ files are generated and quality control measures are performed on raw and aligned reads for evaluation by internal staff to ensure they meet minimum metrics
- RUO Standard Analysis Pipeline
- FASTQ files are aligned to the GRCH38 genomic reference
- Multiple lanes of sequencing are merged and then pre-processed to mark duplicate, then normalized the base quality scores (call ready alignments)
- If the case Whole Genome sequencing available from a corresponding tumour with normal; the call ready alignement is used for detection of Minimal Residual Disease (MRD)
- The RUO assay is available at 30X coverage
- The Clinical assay pricing includes extraction; RUO pricing does not
- Minimum DNA input requirement as measured by fluorometric quantitations:
- 10 ng (cfDNA from plasma)
- There is a 45 day turnaround time for clinical cases
Targeted Sequencing – REVOLVE Panel (TAR)
This clinical assay is available in two (2) stages: an initial submission of tumour or cfDNA + buffy coat and; a follow-up submission of only tumour or cfDNA.
The KAPA Hyper Prep Kit (Roche) is used to create whole genome libraries, from which target fragments are pulled down using the REVOLVE panel probe set. Libraries are then visualized and quantitated using the Fragment Analyzer and Qubit 4.0. Library quality is determined using shallow MiSeq sequencing to assess mapping to genomic regions. Libraries are then sequenced using 2×150 paired reads on an Illumina NovaSeq X Plus system to the requested target depth. Analysis proceeds through a validated pipeline after sequencing.
- Clinical assay with geneticist approved-report available at:
- 15 000X coverage for tumour
- 5000X for normal samples
- Minimum DNA requirements as measured by fluorometric quantitation:
- 20 ng (cfDNA from plasma)
- 40 ng (genomic DNA from buffy coat)
- 40 ng (genomic DNA from fresh frozen)
- 40 ng (genomic DNA from FFPE)
- There is a 21 day turnaround time for clinical cases
- Pricing includes extraction
Whole Exome Sequencing
The KAPA Hyper Prep Kit (Roche) is used to create whole genome libraries, from which exome target fragments are pulled down using the IDT xGen Exome Research Panel. Initial library prep quality is assessed via visualization of size distribution on a fragment analyzer or TapeStation, and quantitated using a Qubit fluorometer. Library quality is determined using shallow MiSeq sequencing to assess mapping to genomic regions. Libraries are then sequenced using 2×150 paired reads on an Illumina NovaSeq X Plus system to the requested target depth. Analysis proceeds through a validated pipeline after sequencing. FASTQ files called from the sequencing instrument are aligned using BWAmem. Quality control measures are performed on both raw and aligned reads, which are evaluated by internal staff to ensure they meet minimum metrics.
- RUO assay (data only) available in 100X or 200X coverage options
- Minimum DNA input requirements as measured by fluorometric quantitation:
- 230 ng (genomic DNA from buffy coat)
- 80 ng (genomic DNA from fresh frozen)
- 130 ng (genomic DNA from FFPE)
cfMeDIP Sequencing
The KAPA Hyper Prep Kit (Roche) is used to create whole genome libraries, from which methylated fragments are immunoprecipitated and pulled down using the Diagenode-MagMeDIP Kit. Initial library prep quality is assessed via visualization of size distribution on a Fragment Analyzer or TapeStation, and quatitated using a Qubit fluorometer. Library quality is determined using shallow MiSeq sequencing to asses mapping to genomic regions. Libraries are then sequenced using 2×150 paired reads on an Illumina NovaSeq X Plus system to the requested target depth. Analysis proceeds through a validated pipeline after sequencing. FASTQ files called from the sequencing instrument are aligned using Bowtie2. Quality control measures are perfomed on both the raw and aligned reads, which are evaluated by internal staff to ensure they meet minimum metrics.
- RUO assay (data only) is available in 120M PE reads coverage
- Minimum DNA input requirements as measured by fluorometric quantitation
- 10 ng (cfDNA from plasma)
Enzymatic Methyl-Seq
The NEBNext Enzymatic Methyl-seq kit is used to create whole genome DNA methylation libraries. Initial library prep quality is assessed via visualization of size distribution on a Fragment Analyzer or TapeStation, and quantitated using a Qubit fluorometer. Library quality is determined using shallow MiSeq sequencing to assess mapping to genomic regions. Libraries are then sequenced using 2X150 paired reads on an Illumina NovaSeq X Plus system to the requested target depth. Analysis proceeds through a standard Quality Control pipeline after sequencing. FASTQ files called from the sequencing instrument are aligned using bwa-meth. Quality control measures are performed on both raw and aligned reads, which are evaluated by internal staff to ensure they meet minimum metrics.
- RUO assay (FASTQ data only) available at 30X coverage/ 400M clusters
- Minimum DNA input as measured by fluorometric quantitation
- 10 ng (cfDNA from plasma)
- 50 ng (genomic DNA from fresh frozen)
- 100 ng (genomic DNA from FFPE)