442e433a90b25deb87f10e6cf1b7b608bb0a6d67 max Sat Sep 26 21:56:06 2026 -0700 sfariSparkWgs45kAsd: flag 25M insertions of non-human (oral bacteria) sequence as FILTER NonHumanIns and hide them by default; add SFARI SPARK 45k WGS to the combined tracks without those insertions and relabel the 12k pilot as SFARI SPARK iWGS v1.1 Pilot, refs #38424 diff --git src/hg/makeDb/trackDb/human/sfariSparkExomes.html src/hg/makeDb/trackDb/human/sfariSparkExomes.html index d22015a1b5f..0d7df3cd6ba 100644 --- src/hg/makeDb/trackDb/human/sfariSparkExomes.html +++ src/hg/makeDb/trackDb/human/sfariSparkExomes.html @@ -1,193 +1,210 @@ <h2>Description</h2> <p> The <a href="https://sparkforautism.org/" target="_blank">Simons Foundation Autism Research Initiative (SFARI)</a> recruited a large cohort of families with autistic children who provided DNA samples and phenotypes. 54,558 families, parents and their children were sequenced, a total of 142,357 individuals with whole-exome (WES) and 12,519 with whole-genome sequencing (WGS). The data contains 32,559 trios and 8,895 quads (one sibling without autism), and 824 twins. </p> <p> The <a href="https://base.sfari.org/dataset/DS0000135" target="_blank">SPARK WGS August 2026 release</a> (SFARI Base dataset DS0000135) adds whole genomes for 45,178 individuals from 21,003 families, 20,858 of them with autism. This is a mostly new set of people: only 14 of them are also in the 12,519-genome release and 201 in the exome release. It includes about 4,900 trios with an autistic child and 1,800 quads. The genomes were sequenced PCR-free on the Illumina NovaSeq X at Broad Clinical Labs. The track "SFARI SPARK 45k WGS ASD" shows this release. Its counts were computed from the genotypes, with the autism/non-autism split -and all variants, including those seen only once (see Methods). +and all variants, including those seen only once. Millions of long insertions in this release +are bacterial DNA from the saliva samples, not human variants. They are marked and hidden by +default (see Methods). </p> <p> The same frequencies shown here are also available publicly on the <a href="https://genomes.sfari.org/" target="_blank">SFARI Genome Browser</a>. See (SPARK et al, Neuron 2018) for details. </p> <h3>Phenotype-stratified counts</h3> <p> In addition to the overall allele count (AC), allele number (AN), and allele frequency (AF), each variant record carries counts split by autism status (the <tt>asd</tt> column of the SPARK individual registration file): </p> <ul> <li><tt>AC_AUT</tt>, <tt>AN_AUT</tt>, <tt>AF_AUT</tt> — individuals coded as autistic (<tt>asd=TRUE</tt>).</li> <li><tt>AC_NON_AUT</tt>, <tt>AN_NON_AUT</tt>, <tt>AF_NON_AUT</tt> — individuals coded as non-autistic (<tt>asd=FALSE</tt>); these are mostly parents and unaffected siblings of the probands.</li> </ul> <p> A small minority of samples have a blank <tt>asd</tt> value and so contribute only to the overall AC/AN/AF, not to either group total. </p> <h2>Data Access</h2> <p> Due to license restrictions, the data for this track cannot be downloaded from the UCSC Genome Browser. The Table Browser, Data Integrator, and download server are not available for this track. </p> <p> Allele frequencies can also be displayed on the <a href="https://genomes.sfari.org/" target="_blank">SFARI Genome Browser</a>. Full CRAMs and VCFs with genotypes are available from <a href="https://base.sfari.org/" target="_blank">SFARI Base</a>. They require a data access request, which is usually reviewed quickly. More information is available in the <a href="https://cohorts-cdn.simonsfoundation.org/spark/researcher_packets/SPARK_SFARI_Researcher_Welcome_Packet.pdf" target="_blank">SPARK Welcome Packet</a>. </p> <h2>Methods</h2> <p>The genome browser track project was approved by the Simons Foundation under request number 14584.1. The multi-sample project VCFs (pVCFs) for the 2024 WES and the 12,519-genome WGS releases were downloaded from <a href="https://base.sfari.org/" target="_blank">SFARI Base</a> using Globus. No minimum allele frequency cutoff was applied.</p> <p> Because the genotype-level pVCFs cannot be redistributed, they were reduced to anonymous, sites-only VCFs carrying only the overall allele count (AC), allele number (AN) and frequency (AF), plus the autism-status counts described above, with the bcftools <a href="http://samtools.github.io/bcftools/howtos/plugin.fill-tags.html" target="_blank">fill-tags</a> plugin (its <tt>-S</tt> option produces the ASD/non-ASD splits), then normalized. The variants are also annotated with predicted protein consequences using <a href="http://samtools.github.io/bcftools/bcftools.html#csq" target="_blank">bcftools csq</a> against Ensembl gene models; that annotation is displayed in the combined frequency tracks of this collection. The exact commands for both steps are in the makeDoc file linked below.</p> <p> The track "SFARI SPARK 45k WGS ASD" was made from the genotype-level pVCFs of the WGS August 2026 release, downloaded from SFARI Base with Globus. They are split into 2.5 Mb chunks. The samples were grouped by the <tt>asd</tt> column of the release's sample metadata file: 20,858 autistic and 24,320 non-autistic individuals, with no sample left unassigned. As for the older SPARK tracks, <tt>bcftools +fill-tags</tt> computed AC, AN and AF overall and per group from the called genotypes. Missing genotypes do not count towards AN. The genotypes were then removed. Multiallelic records were split and left-aligned with <tt>bcftools norm</tt>. Alleles that no individual carries after joint genotyping (AC=0) were removed. There is no allele count cutoff. Records that GLnexus marks with the filter <tt>MONOALLELIC</tt> are alleles it could not merge into an overlapping site. In these, only the carriers have a genotype, so AN would count only them and AF would be 1. For these records, AN was set to twice the number of individuals in each group and AF was recomputed from AC. The INFO field <tt>VARLEN</tt> is the length of ALT minus the length of REF: positive for insertions, negative for deletions, 0 for substitutions. The pVCFs were processed in 500 kb pieces on our compute cluster. The scripts are <tt>sparkWgs45kPvcfToSites.sh</tt>, <tt>sparkWgs45kPvcfJobs.sh</tt> and <tt>sparkWgs45kPvcfMerge.sh</tt>. </p> +<p> +This release contains millions of long insertions whose inserted sequence is DNA from +bacteria that live in the mouth, such as <em>Streptococcus mitis</em> and <em>Neisseria +subflava</em>. The DNA was extracted from saliva. We think that reads which are partly +bacterial were soft-clipped by the aligner, and the variant caller then turned the bacterial +part into an insertion. To find them, every inserted sequence of 20 bp or longer was aligned +to the human genome with <tt>bwa mem</tt>. An insertion whose sequence aligns over less than +80% of its length, or with more than 10% mismatches, and which is not also in the gnomAD v4.1 +genomes, gets the value <tt>NonHumanIns</tt> in the FILTER column. These insertions are hidden +by default; to show them, uncheck NonHumanIns under "Exclude variants with these FILTER +values" on this configuration page. The gnomAD check keeps real insertions that are missing +from the reference genome, some of which are common. The script is +<tt>sparkWgs45kFlagNonHumanIns.sh</tt>. +</p> + <p>The sequencing and variant-calling methods are documented as follows by SFARI:</p> <ul> <li> <b>WGS, August 2026 release</b>: DNA was extracted from saliva at Broad Clinical Labs or Prevention Genetics. Genomes were sequenced PCR-free on the Illumina NovaSeq X at Broad Clinical Labs. Samples were kept if contamination was 2.5% or less and 75% or more of reads were mapped. Genetic relationships were checked against the SPARK pedigrees. Reads were realigned to GRCh38 with BWA-MEM2 v2.3, duplicates were marked with GATK MarkDuplicates, and per-sample gVCFs were called with GATK HaplotypeCaller v4.6.2.0. All gVCFs were then joint-called with GLnexus v1.4.1 using its GATK configuration. </li> <li> <b>WGS, 12,519 genomes</b>: This release consists of sequence and variant call data for 12,519 unique individuals, of which 12,517 (99.98%) have available genome-wide SNP genotype data. Sequencing and genotyping of all samples in this release was performed at New York Genome Center (NYGC). DNA from saliva samples were extracted and prepared with PCR-free methods and sequenced with paired-end sequencing of 150 bases on the Illumina NovaSeq 6000 system. Alignment of reads to the human reference genome version GRCh38, duplicate read marking, and Base Quality Score Recalibration (BQSR) were performed by New York Genome Center (NYGC). Whole-genome sequencing data were processed using a standardized, functionally equivalent CCDG pipeline with alignment to the GRCh38DH (1000 Genomes) reference using BWA-MEM v0.7.15 (deterministic settings, no -M, use of .alt contigs), Picard-equivalent duplicate marking (Picard ≥2.4.1 or equivalent), no indel realignment, and base quality score recalibration with GATK (dbSNP138, Mills and 1000G gold-standard indels, known indels). Final outputs were stored as lossless CRAM files with complete SAM-compliant read-group annotations and mandatory 4-bin base-quality compression (Q2—6, 10, 20, 30), and all implementations were validated for functional equivalence across centers before use. Variant Calling was performed using DeepVariant. See <a href="https://github.com/CCDG/Pipeline-Standardization/blob/master/PipelineStandard.md" target="_blank">CCDG pipeline details</a>. </li> <li> <b>WES</b>: This release contains sequence data for 142,357 individuals and genotyping data for 141,368 individuals. DNA was sequenced from saliva for all samples and all participants consented to having their genetic data shared by Regeneron. Exomes for all samples were sequenced with short-read, paired-end sequencing of 150 bases on Illumina NovaSeq 6000 machines using S2/S4 flow cells. Sequencing and genotyping was performed across nine batches (WES1 through WES9) at the Regeneron Genetics Center (RGC) and integrated together for this data release. All sequencing batches were processed using the same DNA extraction methods and sequencing machines, however two different exome capture panels were used, as described below. Genotyping was performed using a SNP genotyping array for WES1 through WES4 and using "genotyping-by-sequencing" (GxS) for WES5 through WES9. The first four sequencing batches were sequenced at Regeneron using custom NEB/Kapa reagents with the IDT (Integrated DNA Technologies) xGen capture platform, including custom exome capture regions. Samples starting with batch WES5 were sequenced using the Twist Bioscience Human Comprehensive Exome panel, combined with spike-ins for sequencing genotyping sites (see Genotyping Methods), the full mitochondrial genome, and coverage boosted at selected sites for assaying clonal hematopoiesis of indeterminate potential (CHIP). SFARI performed SNV/indel calling via DeepVariant and GATK to generate gVCFs, pairwise relatedness inferred using PLINK v1.9 IBD estimates from common SNPs (AF ≥ 0.01, dbSNP v151) with ≥15% relatedness flagged, and comprehensive individual- and family-level quality control executed using the internal GenomeCheckMate pipeline to exclude samples based on contamination (≥5%), insufficient coverage (<20x in <80% of targets), sex discordance, pedigree/IBD inconsistencies, unregistered relationships, unexpected duplicates, or excess relatedness, after which QC-passing individuals (selecting the most recent passing sample per person) were retained for variant calling and joint genotyping. </li> </ul> <p> The complete, runnable command history for downloading, counting and annotating the SFARI data, alongside every other cohort in this collection, is in the <a href="https://github.com/ucscGenomeBrowser/kent/blob/master/src/hg/makeDb/doc/hg38/varFreqs.txt" target="_blank">makeDoc file</a>; search it for "SFARI SPARK". The scripts it calls, including <tt>sparkMergeVcfAddCounts.sh</tt> (allele counts), <tt>sparkWgs45kPvcfToSites.sh</tt> (45k WGS genotype counts) and <tt>mergeAndAnnotate.sh</tt> (merge plus consequence annotation), are in the <a href="https://github.com/ucscGenomeBrowser/kent/tree/master/src/hg/makeDb/scripts/varFreqs" target="_blank">varFreqs scripts directory</a>. </p> <h2>References</h2> <p> SPARK Consortium. Electronic address: <A HREF="mailto:pfeliciano@simonsfoundation.org">pfeliciano@simonsfoundation.org</A><!-- above address is pfeliciano at simonsfoundation.org -->, SPARK Consortium. <a href="https://linkinghub.elsevier.com/retrieve/pii/S0896-6273(18)30018-7" target="_blank"> SPARK: A US Cohort of 50,000 Families to Accelerate Autism Research</a>. <em>Neuron</em>. 2018 Feb 7;97(3):488-493. PMID: <a href="https://www.ncbi.nlm.nih.gov/pubmed/29420931" target="_blank">29420931</a>; PMC: <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444276/" target="_blank">PMC7444276</a> </p>