How to read the Alzheimer’s gene panel reports
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We use the PiVAT variant reporting tool from Pillar Biosciences to analyze genotyping results from Alzheimer’s gene panels. PiVAT generates a set of report files that are stored in the “Genotype” directory in your Box.com account.
Alzheimer’s Gene Panel Report Files
Here is a list of files in the “Genotype” directory.
| File Name | Description |
|---|---|
| sampleName-P_PiVATRESULTS_2020.1.2.xlsx | Here is a sample report for the Alzheimer's gene panel. This is perhaps the most important file for reviewing genotype results. Descriptions and definitions for the report table columns are shown here. |
| sampleName-P_PiVATVCF_2020.1.2.vcf | Variant Call Format file that stores gene variant information. |
| sampleName-P.bam | Binary Alignment Map file. |
| sampleName-P.bam.bai | Indexed version of the sampleName-P.bam file. |
| sampleName-P_PiVATBAM_2020.1.2.bam | Same as the file sampleName-P.bam but designed for PiVAT internal use. |
| sampleName-P_PiVATBAM_2020.1.2.bam.bai | Same as the file sampleName-P.bam.bai but designed for PiVAT internal use. |
| samtools.log | Log file from the SAMtools bioinformatics suite. |
| sampleName-P_PiVATRESULTSRD_2020.1.2.p | System generated file that you can ignore. |
The primary analysis file “sampleName-P_PiVATRESULTS_2020.1.2.xlsx” includes two tabs as follows:
- Final Annotated Variants. This tab shows nucleotide variants and mutations (i.e. single nucleotide polymorphisms, insertions, deletions, etc.) in the sample sequence compared to the reference human genome. This tab typically includes the affected gene name(s) and consequences of the mutation(s).
- Final False Positive Variants. This tab is included in the results to show possible variants and mutations that did not pass the variant calling algorithms minimum thresholds. These mutations may warrant further consideration.
Alzheimer's Gene Panel Variant Report Descriptions
| Name | Description |
|---|---|
| Sample_ID | User defined library ID, derived from FASTQ file name |
| Chromosome | Chromosome number in human genome where variant occurs |
| Position | Base position on given chromosome where variant occurs (hg19) |
| REF | Sequence found in human reference genome (hg19) |
| ALT | Variant sequence found in sample |
| Location | Genomic coordinates of variant (hg19) |
| Variant_Type | SNV=single nucleotide variant; Deletion=one or more base deletion; Insertion=one or more base insertion; Delins="deletion-insertion", variant characterized by both a deletion of one or more bases AND an insertion of one of more bases on the same allele. i.e. AGACTA ---> ATTCTA resulting from deletion of GA and insertion of TT or AGACTA ---> ATCTA resulting from deletion of GA and insertion of T |
| Variant_Length | Number of affected bases |
| Amplicon_ID | Amplicon in assay in which variant occurs |
| Variant_Read_Frequency_(%) | Occurrence of variant in sequencing data, as a percentage of total sequenced reads in the given segment. (Variant_Coverage / Total_Coverage)*100 |
| Variant_Coverage | Number of reads that contain variant in segment |
| Total_Coverage | Total number of reads in segment |
| Variant_Quality | Measure of variant call quality. Score is from 0 (low quality) to 40 (highest quality) |
| Variant_Read_Direction_Ratio | Measure of strand bias in variant calling. Value of 0 indicates variant was found on both the positive strand and negative strand at equal rates (ideal). Values greater than zero or less than zero indicate percentage of bias towards the negative strand (negative values) or positive strand (positive values) |
| Zygosity | Germline mutation panels only. HETEROZYGOUS=one out of two alleles contain variant; HOMOZYGOUS=both alleles contain variant |
| Consequence | Type of mutation. Examples: frameshift_variant=variant produces an mRNA transcript that is out of the normal reading frame. synonymous_variant=variant does not affect amino acid sequence of final protein due to redundancy of codons. missense_variant=variant leads to change in amino acid sequence. inframe_deletion=deletions of 3, or multiples of 3, which remove whole codon sequences. intron_variant=variant occurs in intron. splice_region_variant=variant in splice site, may lead to splicing error in final mRNA transcript. stop_gained=variant results in premature stop codon in mRNA transcript. 3_prime_UTR_variant=variant occurs in 3" untranslated region (UTR). |
| Impact | Impact of variant on normal gene function |
| Gene_Symbol | Gene name |
| Gene_ID | NCBI gene ID number |
| Feature | NCBI transcript identifier |
| HGVSC | HGVSC variant name. Describes position and variant change in gene (nucleic acid) sequence in the Feature |
| HGVSP | HGVSP variant name. Describes position and variant change in protein (amino acid) sequence in the Feature |
| Exon | Affected exon number in Feature out of total number of exons. Applies to variants within exon regions only. (affected exon # / total # of exons) |
| Intron | Affected intron number in Feature out of total number of introns. Applies to variants within intron regions only. (affected intron # / total # of introns) |
| CDS_Position | cDNA sequence position in Feature |
| Protein_Position | Affected amino acid number in Feature |
| Amino_Acids | Amino acid resulting from variant (reference amino acid / variant amino acid ) |
| Codons | Position in codon where variant occurs and resulting codon with variant |
| Co-located_Known_Variation | Variants that occur at the same position in publicly availabledatabases |
| Strand Transcribed strand. | -1=negative strand, 1=postive strand |
| Repeat | Number of repeat bases, including repeat sequence (if applicable). i.e. 14G indicates a repeat of 14 Gs. |
| HGVS_Offset | Correction factor to synchronize genomic base position for indels in repeat regions in negative strand genes. Read alignment uses left align paradigm (left most base assumed to be indel; first genomic position) while HGVS uses right-align paradigm (right most base assumed to be indel; first cDNA position). |
| SIFT | Output from SIFT (v5.2.2). PREDICTION(SIFT score). Predicts impact of variant on protein function. Prediction possibilities: tolerated, deleterious. SIFT score is a range from 0 - 1. Scores > 0.05 are tolerated, scores of <0.05 are deleterious. |
| POLYPHEN | Output from PolyPhen (v2.2.2). PREDICTION(PolyPhen score). Predicts impact of variant on protein function. Prediction Overall Stats Tab possibilities: benign, possibly damaging, probably damaging. PolyPhen score is a range from 0 (benign) - 1 (probably damaging) and is the probability of variant leading to a damaging mutation. Benign=variant unlikely to cause damage to protein function. possibly damaging=variant likely to cause damage to protein function but prediction is with low confidence. probably damaging=variant likely to cause damage to protein function and prediction is with high confidence. |
| AF | Allele frequency of variant found in global population (1000 Genomes Phase 3, dbSNP v147) |
| AFR_AF | Allele frequency of variant found in African populations (dbSNP v147) |
| AMR_AF | Allele frequency of variant found in American populations (dbSNP v147) |
| EAS_AF | Allele frequency of variant found in East Asian populations (dbSNP v147) |
| EUR_AF | Allele frequency of variant found in European populations (dbSNP v147) |
| SAS_AF | Allele frequency of variant found in South Asian populations (dbSNP v147) |
| CLIN_SIG | Clinical significance (ClinVar v201610) |
| Statistic | Description |
| Total Reads | Total number of sequencing reads for the library |
| Overall:Q=30 | Percentage of bases with Q score greater than or equal to 30 |
| Overall:Q=20 | Percentage of bases with Q score greater than or equal to 20 |
| Properly Paired Read | Number of read mates properly paired, from paired end sequencing |
| Properly Paired Read (%) | Percentage of total reads properly paired |
| Mapped Reads | Percentage of total reads that map to human genome (hg19) |
| Mapping Rate (%) | Percentage of total reads that map to human genome (hg19) |
| On Target Reads | Reads that map to target amplicon regions of interest (ROIs) |
| On Target Rate (%) | Percentage of mapped reads that map to target amplicon regions of interest (ROIs) |
| Insert Size Mean | Mean size of the library insert (varies based on panel) |
| Insert Size Median | Median size of the library insert |
| Insert Size Std Dev | Standard deviation of library insert size |
| Coverage_Mean | Mean base coverage of all bases within the de/ned ROI; with paired end sequencing, merged paired-end reads (forward and reverse) create a coverage of 1x |
| STDEV | Standard deviation of mean base coverage |
| Coverage_Median | Median base coverage of all bases within the de/ned ROI; with paired end sequencing, merged paired-end reads (forward and reverse) create a coverage of 1x |
| Coverage_Max | Maximum base coverage of all bases within the de/ned ROI |
| Coverage_Min | Minimum base coverage of all bases within the de/ned ROI |
| Base_Coverage_Depth_>_(Nx) | Percent of bases that have a minimum base coverage greater than or equal to Nx (absolute coverage) |
| Base_Coverage_Depth_>_(Nx)_Relative_to_Mean_Coverage | Percent of bases that obtain at least Nx*mean base coverage, usually described as percent of mean base coverage. Used to determine uniformity of base coverage across ROIs in panel. Value of 100 indicates 100% of bases in all ROI are above the given Nx relative to the Coverage_Mean. |
If you need additional assistance with the Alzheimer’s report files, please contact us.
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