Screening

Germline variant discovery for CoGenesis® neuro targeted gene panel v3. The coding regions, splice sites, selected promoters and introns of selected genes were sequenced using with 150bp paired-end reads on the Illumina Nextseq 500 platform or the Illumina Nextseq 2000 platform at a sequencing depth of >150X. The DNA sequence … Germline variant discovery for CoGenesis® neuro targeted gene panel v3. The coding regions, splice sites, selected promoters and introns of selected genes were sequenced using with 150bp paired-end reads on the Illumina Nextseq 500 platform or the Illumina Nextseq 2000 platform at a sequencing depth of >150X. The DNA sequence was mapped to, and analyzed in comparison with, the published human genome build (GRCh38 reference sequence). The targeted coding exons and splice junctions of the known protein-coding RefSeq/Ensembl genes were assessed for the average depth of coverage and data quality threshold values. View more

None/not applicable

Raw reads were first processed using quality-check programs, such as Trimmomatic, to perform quality trimming and remove adapter contaminants. The short-read sequences were aligned to the human genome reference (version GRCh38) using BWA-MEM (Li and Durbin, 2009), followed by PCR duplicates marking, local realignment around indels, and base quality score … Raw reads were first processed using quality-check programs, such as Trimmomatic, to perform quality trimming and remove adapter contaminants. The short-read sequences were aligned to the human genome reference (version GRCh38) using BWA-MEM (Li and Durbin, 2009), followed by PCR duplicates marking, local realignment around indels, and base quality score recalibration using Picard and Genome Analysis Toolkit (McKenna et al., 2010). The calling of SNPs and small indels was performed using batch genotype calling of all subjects with GATK HaplotypeCaller, which outputs variant sites that can be observed in at least one subject. The variants were fltered according to the GATK best practices, followed by minor allele frequency (MAF) fltering according to population genetics databases (1000 Genome phase 3, GnomAD Exome, NHLBI Exome Sequencing Project 6500;MAF <0.01), except those with previously reported pathogenicity in ClinVar. Copy number variations (CNVs) were called using GATK-CNV and CNVkit (Talevich et al., 2016). Structural variations (SVs) were called using Manta (Chen et al. 2016). Short tandem repeat (STR) variations were called using inhouse algorithm as described in (Yu et al., 2019) and GangSTR (Nima et al. 2019). Detected pathogenic variations were confirmed by Sanger sequencing, if applicable. View more

Tests performed
Entire test performed in-house

The concordance rate of the test to WES is 99.94% for SNV detection and 93.2% for indel detection. The test has reached a specificity of 0.88 and a sensitivity of 0.82 in our SCA3 cohort in short tandem repeat detection.

The interpretation of sequence variants is based on the current understanding of the variant at the time it was observed, which may change over time as more information about the genes becomes available. Not all variants are represented in this report. Absence of a plausible explanation for the reported phenotype … The interpretation of sequence variants is based on the current understanding of the variant at the time it was observed, which may change over time as more information about the genes becomes available. Not all variants are represented in this report. Absence of a plausible explanation for the reported phenotype by targeted sequencing does not exclude a genetic basis of the patient’s condition. Some types of genetic abnormalities, such as copy number changes, may not be detectable with the technologies performed by this targeted analysis test. It is possible that the genomic region where a disease causing mutation exists in the individual was not captured using the current technologies and therefore was not detected. Additionally, it is possible that a particular genetic abnormality may not be recognized as the underlying cause of the genetic disorder due to incomplete scientifc knowledge about the function of all genes in the human genome and the impact of variants in those genes. Only variants in genes associated with the medical condition, or thought to be clinically relevant potentially for the individual’s medical condition, are reported here. View more

Is proficiency testing performed for this test? Help
Yes

Method used for proficiency testing: Help
EQA

PT Provider: Help
Genomic Quality Assessment (GenQA)

Description of PT method: Help
Testing one DNA sample using any NGS platform/panel performed in the diagnostic laboratory for germline testing

Description of internal test validation method: Help
Manufacturer defines performance specification and we will vertify the defined performance specification, including the base quality score, coverage and variant quality.

Software used to interpret novel variations Help
In-house software