A human-specific VNTR in the TRIB3 promoter causes gene expression variation between individuals.

Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that human TRIB3 promoter contains a 33-bp variable number tandem repeat (VNTR) and characterize the heterogeneity and function of this genetic element. Analysis of human populations around the world uncovered the existence of alleles ranging from 1 to 5 copies of the repeat, with 2-, 3- and 5-copy alleles being the most common but displaying considerable geographical differences in frequency. The repeated sequence overlaps a C/EBP-ATF transcriptional regulatory element and is highly conserved, but not repeated, in various mammalian species, including great apes. The repeat is however evident in Neanderthal and Denisovan genomes. Reporter plasmid experiments in human cell culture reveal that an increased copy number of the TRIB3 promoter 33-bp repeat results in increased transcriptional activity. In line with this, analysis of whole genome sequencing and RNA-Seq data from human cohorts demonstrates that the copy number of TRIB3 promoter 33-bp repeats is positively correlated with TRIB3 mRNA expression level in many tissues throughout the body. Moreover, the copy number of the TRIB3 33-bp repeat appears to be linked to known TRIB3 eQTL SNPs as well as TRIB3 SNPs reported in genetic association studies. Taken together, the results indicate that the promoter 33-bp VNTR constitutes a causal variant for TRIB3 expression variation between individuals and could underlie the results of SNP-based genetic studies.

KATK: Fast genotyping of rare variants directly from unmapped sequencing reads.

KATK is a fast and accurate software tool for calling variants directly from raw next-generation sequencing reads. It uses predefined k-mers to retrieve only the reads of interest from the FASTQ file and calls genotypes by aligning retrieved reads locally. KATK does not use data about known polymorphisms and has NC (no call) as the default genotype. The reference or variant allele is called only if there is sufficient evidence for their presence in data. Thus it is not biased against rare variants or de-novo mutations. With simulated datasets, we achieved a false-negative rate of 0.23% (sensitivity 99.77%) and a false discovery rate of 0.19%. Calling all human exonic regions with KATK requires 1-2 h, depending on sequencing coverage.

A recent bottleneck of Y chromosome diversity coincides with a global change in culture.

It is commonly thought that human genetic diversity in non-African populations was shaped primarily by an out-of-Africa dispersal 50-100 thousand yr ago (kya). Here, we present a study of 456 geographically diverse high-coverage Y chromosome sequences, including 299 newly reported samples. Applying ancient DNA calibration, we date the Y-chromosomal most recent common ancestor (MRCA) in Africa at 254 (95% CI 192-307) kya and detect a cluster of major non-African founder haplogroups in a narrow time interval at 47-52 kya, consistent with a rapid initial colonization model of Eurasia and Oceania after the out-of-Africa bottleneck. In contrast to demographic reconstructions based on mtDNA, we infer a second strong bottleneck in Y-chromosome lineages dating to the last 10 ky. We hypothesize that this bottleneck is caused by cultural changes affecting variance of reproductive success among males.

DOCEST-fast and accurate estimator of human NGS sequencing depth and error rate.

Motivation: Accurate estimation of next-generation sequencing depth of coverage is needed for detecting the copy number of repeated elements in the human genome. The common methods for estimating sequencing depth are based on counting the number of reads mapped to the genome or subgenomic regions. Such methods are sensitive to the mapping quality. The presence of contamination or the large deviance of an individual genome from the reference may introduce bias in depth estimation. Results: Here, we present an algorithm and implementation for estimating both the sequencing depth and error rate from unmapped reads using a uniquely filtered k-mer set. On simulated reads with 20× coverage, the margin of error was less than 0.01%. At 0.01× coverage and the presence of 10-fold contamination, the precision was within 2% for depth and within 10% for error rate. Availability and implementation: DOCEST program and database can be downloaded from https://bioinfo.ut.ee/docest/. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

AluMine: alignment-free method for the discovery of polymorphic Alu element insertions.

BACKGROUND: Recently, alignment-free sequence analysis methods have gained popularity in the field of personal genomics. These methods are based on counting frequencies of short k-mer sequences, thus allowing faster and more robust analysis compared to traditional alignment-based methods. RESULTS: We have created a fast alignment-free method, AluMine, to analyze polymorphic insertions of Alu elements in the human genome. We tested the method on 2,241 individuals from the Estonian Genome Project and identified 28,962 potential polymorphic Alu element insertions. Each tested individual had on average 1,574 Alu element insertions that were different from those in the reference genome. In addition, we propose an alignment-free genotyping method that uses the frequency of insertion/deletion-specific 32-mer pairs to call the genotype directly from raw sequencing reads. Using this method, the concordance between the predicted and experimentally observed genotypes was 98.7%. The running time of the discovery pipeline is approximately 2 h per individual. The genotyping of potential polymorphic insertions takes between 0.4 and 4 h per individual, depending on the hardware configuration. CONCLUSIONS: AluMine provides tools that allow discovery of novel Alu element insertions and/or genotyping of known Alu element insertions from personal genomes within few hours.

Genetic variation in the Estonian population: pharmacogenomics study of adverse drug effects using electronic health records.

Pharmacogenomics aims to tailor pharmacological treatment to each individual by considering associations between genetic polymorphisms and adverse drug effects (ADEs). With technological advances, pharmacogenomic research has evolved from candidate gene analyses to genome-wide association studies. Here, we integrate deep whole-genome sequencing (WGS) information with drug prescription and ADE data from Estonian electronic health record (EHR) databases to evaluate genome- and pharmacome-wide associations on an unprecedented scale. We leveraged WGS data of 2240 Estonian Biobank participants and imputed all single-nucleotide variants (SNVs) with allele counts over 2 for 13,986 genotyped participants. Overall, we identified 41 (10 novel) loss-of-function and 567 (134 novel) missense variants in 64 very important pharmacogenes. The majority of the detected variants were very rare with frequencies below 0.05%, and 6 of the novel loss-of-function and 99 of the missense variants were only detected as single alleles (allele count = 1). We also validated documented pharmacogenetic associations and detected new independent variants in known gene-drug pairs. Specifically, we found that CTNNA3 was associated with myositis and myopathies among individuals taking nonsteroidal anti-inflammatory oxicams and replicated this finding in an extended cohort of 706 individuals. These findings illustrate that population-based WGS-coupled EHRs are a useful tool for biomarker discovery.

SNPmasker: automatic masking of SNPs and repeats across eukaryotic genomes.

SNPmasker is a comprehensive web interface for masking large eukaryotic genomes. The program is designed to mask SNPs from recent dbSNP database and to mask the repeats with two alternative programs. In addition to the SNP masking, we also offer population-specific substitution of SNP alleles in genomic sequence according to SNP frequencies in HapMap Phase II data. The input to SNPmasker can be defined in chromosomal coordinates or inserted as a sequence. The sequences masked by our web server are most useful as a preliminary step for different primer and probe design tasks. The service is available at http://bioinfo.ebc.ee/snpmasker/ and is free for all users.

FastGT: an alignment-free method for calling common SNVs directly from raw sequencing reads.

We have developed a computational method that counts the frequencies of unique k-mers in FASTQ-formatted genome data and uses this information to infer the genotypes of known variants. FastGT can detect the variants in a 30x genome in less than 1 hour using ordinary low-cost server hardware. The overall concordance with the genotypes of two Illumina "Platinum" genomes is 99.96%, and the concordance with the genotypes of the Illumina HumanOmniExpress is 99.82%. Our method provides k-mer database that can be used for the simultaneous genotyping of approximately 30 million single nucleotide variants (SNVs), including >23,000 SNVs from Y chromosome. The source code of FastGT software is available at GitHub (https://github.com/bioinfo-ut/GenomeTester4/).

MultiPLX: automatic grouping and evaluation of PCR primers.

UNLABELLED: MultiPLX is a new program for automatic grouping of PCR primers. It can use many different parameters to estimate the compatibility of primers, such as primer-primer interactions, primer-product interactions, difference in melting temperatures, difference in product length and the risk of generating alternative products from the template. A unique feature of the MultiPLX is the ability to perform automatic grouping of large number (thousands) of primer pairs. AVAILABILITY: Binaries for Windows, Linux and Solaris are available from http://bioinfo.ebc.ee/download/. A graphical version with limited capabilities can be used through a web interface at http://bioinfo.ebc.ee/multiplx/. The source code of the program is available on request for academic users. CONTACT: maido.remm@ut.ee.

A first-generation linkage disequilibrium map of human chromosome 22.

DNA sequence variants in specific genes or regions of the human genome are responsible for a variety of phenotypes such as disease risk or variable drug response. These variants can be investigated directly, or through their non-random associations with neighbouring markers (called linkage disequilibrium (LD)). Here we report measurement of LD along the complete sequence of human chromosome 22. Duplicate genotyping and analysis of 1,504 markers in Centre d'Etude du Polymorphisme Humain (CEPH) reference families at a median spacing of 15 kilobases (kb) reveals a highly variable pattern of LD along the chromosome, in which extensive regions of nearly complete LD up to 804 kb in length are interspersed with regions of little or no detectable LD. The LD patterns are replicated in a panel of unrelated UK Caucasians. There is a strong correlation between high LD and low recombination frequency in the extant genetic map, suggesting that historical and contemporary recombination rates are similar. This study demonstrates the feasibility of developing genome-wide maps of LD.