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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 NameDescription
sampleName-P_PiVATRESULTS_2020.1.2.xlsxHere 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.vcfVariant Call Format file that stores gene variant information.
sampleName-P.bamBinary Alignment Map file.
sampleName-P.bam.baiIndexed version of the sampleName-P.bam file.
sampleName-P_PiVATBAM_2020.1.2.bamSame as the file sampleName-P.bam but designed for PiVAT internal use.
sampleName-P_PiVATBAM_2020.1.2.bam.baiSame as the file sampleName-P.bam.bai but designed for PiVAT internal use.
samtools.logLog file from the SAMtools bioinformatics suite.
sampleName-P_PiVATRESULTSRD_2020.1.2.pSystem 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

NameDescription
Sample_IDUser defined library ID, derived from FASTQ file name
ChromosomeChromosome number in human genome where variant occurs
PositionBase position on given chromosome where variant occurs (hg19)
REFSequence found in human reference genome (hg19)
ALTVariant sequence found in sample
LocationGenomic coordinates of variant (hg19)
Variant_TypeSNV=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_LengthNumber of affected bases
Amplicon_IDAmplicon 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_CoverageTotal number of reads in segment
Variant_QualityMeasure of variant call quality. Score is from 0 (low quality) to 40 (highest quality)
Variant_Read_Direction_RatioMeasure 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)
ZygosityGermline mutation panels only. HETEROZYGOUS=one out of two alleles contain variant; HOMOZYGOUS=both alleles contain variant
ConsequenceType 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).
ImpactImpact of variant on normal gene function
Gene_SymbolGene name
Gene_IDNCBI gene ID number
FeatureNCBI transcript identifier
HGVSCHGVSC variant name. Describes position and variant change in gene (nucleic acid) sequence in the Feature
HGVSPHGVSP variant name. Describes position and variant change in protein (amino acid) sequence in the Feature
ExonAffected exon number in Feature out of total number of exons. Applies to variants within exon regions only. (affected exon # / total # of exons)
IntronAffected intron number in Feature out of total number of introns. Applies to variants within intron regions only. (affected intron # / total # of introns)
CDS_PositioncDNA sequence position in Feature
Protein_PositionAffected amino acid number in Feature
Amino_AcidsAmino acid resulting from variant (reference amino acid / variant amino acid )
CodonsPosition in codon where variant occurs and resulting codon with variant
Co-located_Known_VariationVariants that occur at the same position in publicly availabledatabases
Strand Transcribed strand.-1=negative strand, 1=postive strand
RepeatNumber of repeat bases, including repeat sequence (if applicable). i.e. 14G indicates a repeat of 14 Gs.
HGVS_OffsetCorrection 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).
SIFTOutput 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.
POLYPHENOutput 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.
AFAllele frequency of variant found in global population (1000 Genomes Phase 3, dbSNP v147)
AFR_AFAllele frequency of variant found in African populations (dbSNP v147)
AMR_AFAllele frequency of variant found in American populations (dbSNP v147)
EAS_AFAllele frequency of variant found in East Asian populations (dbSNP v147)
EUR_AFAllele frequency of variant found in European populations (dbSNP v147)
SAS_AFAllele frequency of variant found in South Asian populations (dbSNP v147)
CLIN_SIGClinical significance (ClinVar v201610)
StatisticDescription
Total ReadsTotal number of sequencing reads for the library
Overall:Q=30Percentage of bases with Q score greater than or equal to 30
Overall:Q=20Percentage of bases with Q score greater than or equal to 20
Properly Paired ReadNumber of read mates properly paired, from paired end sequencing
Properly Paired Read (%)Percentage of total reads properly paired
Mapped ReadsPercentage of total reads that map to human genome (hg19)
Mapping Rate (%)Percentage of total reads that map to human genome (hg19)
On Target ReadsReads 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 MeanMean size of the library insert (varies based on panel)
Insert Size MedianMedian size of the library insert
Insert Size Std DevStandard deviation of library insert size
Coverage_MeanMean 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
STDEVStandard deviation of mean base coverage
Coverage_MedianMedian 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_MaxMaximum base coverage of all bases within the de/ned ROI
Coverage_MinMinimum 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_CoveragePercent 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.