SNP calling by sequencing pooled samples

<p>Abstract</p> <p>Background</p> <p>Performing high throughput sequencing on samples pooled from different individuals is a strategy to characterize genetic variability at a small fraction of the cost required for individual sequencing. In certain circumstances some variability estimators have even lower variance than those obtained with individual sequencing. SNP calling and estimating the frequency of the minor allele from pooled samples, though, is a subtle exercise for at least three reasons. First, sequencing errors may have a much larger relevance than in individual SNP calling: while their impact in individual sequencing can be reduced by setting a restriction on a minimum number of reads per allele, this would have a strong and undesired effect in pools because it is unlikely that alleles at low frequency in the pool will be read many times. Second, the prior allele frequency for heterozygous sites in individuals is usually 0.5 (assuming one is not analyzing sequences coming from, <it>e.g.</it> cancer tissues), but this is not true in pools: in fact, under the standard neutral model, singletons (<it>i.e.</it> alleles of minimum frequency) are the most common class of variants because <it>P</it>(<it>f</it>) ∝ 1/<it>f </it>and they occur more often as the sample size increases. Third, an allele appearing only once in the reads from a pool does not necessarily correspond to a singleton in the set of individuals making up the pool, and vice versa, there can be more than one read – or, more likely, none – from a true singleton.</p> <p>Results</p> <p>To improve upon existing theory and software packages, we have developed a Bayesian approach for minor allele frequency (MAF) computation and SNP calling in pools (and implemented it in a program called <monospace>snape</monospace>): the approach takes into account sequencing errors and allows users to choose different priors. We also set up a pipeline which can simulate the coalescence process giving rise to the SNPs, the pooling procedure and the sequencing. We used it to compare the performance of <monospace>snape</monospace> to that of other packages.</p> <p>Conclusions</p> <p>We present a software which helps in calling SNPs in pooled samples: it has good power while retaining a low false discovery rate (FDR). The method also provides the posterior probability that a SNP is segregating and the full posterior distribution of <it>f</it> for every SNP. In order to test the behaviour of our software, we generated (through simulated coalescence) artificial genomes and computed the effect of a pooled sequencing protocol, followed by SNP calling. In this setting, <monospace>snape</monospace> has better power and False Discovery Rate (FDR) than the comparable packages <monospace>samtools</monospace>, <monospace>PoPoolation</monospace>, <monospace>Varscan</monospace> : for <it>N </it>= 50 chromosomes, <monospace>snape</monospace> has power ≈ 35<it>%</it>and FDR ≈ 2.5<it>%</it>. <monospace>snape</monospace> is available at <url>http://code.google.com/p/snape-pooled/</url> (source code and precompiled binaries).</p

CLAPO syndrome: identification of somatic activating PIK3CA mutations and delineation of the natural history and phenotype

[Purpose]: CLAPO syndrome is a rare vascular disorder characterized by capillary malformation of the lower lip, lymphatic malformation predominant on the face and neck, asymmetry, and partial/generalized overgrowth. Here we tested the hypothesis that, although the genetic cause is not known, the tissue distribution of the clinical manifestations in CLAPO seems to follow a pattern of somatic mosaicism.[Methods]: We clinically evaluated a cohort of 13 patients with CLAPO and screened 20 DNA blood/tissue samples from 9 patients using high-throughput, deep sequencing.[Results]: We identified five activating mutations in the PIK3CA gene in affected tissues from 6 of the 9 patients studied; one of the variants (NM_006218.2:c.248T>C; p.Phe83Ser) has not been previously described in developmental disorders.[Conclusion]: We describe for the first time the presence of somatic activating PIK3CA mutations in patients with CLAPO. We also report an update of the phenotype and natural history of the syndrome.This research was supported by the project “Genetics of vascular and lymphatic malformations” financed with funds donated by Asociación Ultrafondo and Villareal FC, cofinanced by project IP-17 from the funding call “Todos Somos Raros” (Telemaraton TVE promoted by Fundación Isabel Gemio, Federación ASEM, and Federación Española de Enfermedades Raras), cofinanced by the Instituto de Salud Carlos III, FEDER FUNDS FIS PI15/01481, and IIS-Fundación Jiménez Díaz UAM Genome Medicine Chair.Peer reviewe