cDNA-detector: detection and removal of cDNA contamination in DNA sequencing libraries

Background Exogenous cDNA introduced into an experimental system, either intentionally or accidentally, can appear as added read coverage over that gene in next-generation sequencing libraries derived from this system. If not properly recognized and managed, this cross-contamination with exogenous signal can lead to incorrect interpretation of research results. Yet, this problem is not routinely addressed in current sequence processing pipelines. Results We present cDNA-detector, a computational tool to identify and remove exogenous cDNA contamination in DNA sequencing experiments. We demonstrate that cDNA-detector can identify cDNAs quickly and accurately from alignment files. A source inference step attempts to separate endogenous cDNAs (retrocopied genes) from potential cloned, exogenous cDNAs. cDNA-detector provides a mechanism to decontaminate the alignment from detected cDNAs. Simulation studies show that cDNA-detector is highly sensitive and specific, outperforming existing tools. We apply cDNA-detector to several highly-cited public databases (TCGA, ENCODE, NCBI SRA) and show that contaminant genes appear in sequencing experiments where they lead to incorrect coverage peak calls. Conclusions cDNA-detector is a user-friendly and accurate tool to detect and remove cDNA detection in NGS libraries. This two-step design reduces the risk of true variant removal since it allows for manual review of candidates. We find that contamination with intentionally and accidentally introduced cDNAs is an underappreciated problem even in widely-used consortium datasets, where it can lead to spurious results. Our findings highlight the importance of sensitive detection and removal of contaminant cDNA from NGS libraries before downstream analysis.

Anatomical variations of cystic duct insertion and their relationship with choledocholithiasis: an MRCP study

Background The frequency of insertion variations of cystic duct (CD) is inconsistent between studies caused to some extent by the way they give the relative frequency of the variations. Moreover, certain insertion variations have been reported to be associated with choledocholithiasis. This study aimed to assess the frequency of CD insertion variations with a comprehensive way of classification in an unselected population in whom MRCP was performed. Moreover, the relationship between the types of variant insertions and choledocholithiasis using MRCP was also assessed. Patients undergoing magnetic resonance cholangiopancreatography (MRCP) were reviewed retrospectively by two radiologists who were blinded to the clinical data. The normal insertion was defined as the union through middle one third of the lateral border of the extrahepatic bile duct. The transverse site (lateral, medial, anterior, and posterior) and the craniocaudal level (high, mid and low) of insertions and their intersections were assessed using axial and coronal slices, respectively. In addition, the frequencies of the CD insertion variations were compared between choledocholithiasis and control (no-choledocholithiasis) groups. Results A total of 307 patients (124 with choledocholithiasis and 183 controls) were analyzed. A true variant insertion was found in 149 (48.5%) cases. The insertion variations were less frequent in the choledocholithiasis group [50 (40.3%) vs. 99 (54.1%), respectively, p = 0.018]. The frequencies of craniocaudal level of insertion differed significantly between groups (p = 0.014) that was driven by a lower rate of low medial insertion (1.6% vs. 9.8%, respectively) in the choledocholithiasis group. The frequencies of transverse site of insertion were similar between groups (p = 0.314). The low medial insertion was 80.7% less likely associated with choledocholithiasis even after adjustment for age (Odds ratio: 0.193, 95% Confidence interval: 0.039–0.954, p = 0.044). The interreader agreement for insertion assessment was good (Cohen’s Kappa: 0.748, p < 0.001). Conclusions The prevalence of CD insertion variations in an unselected population undergoing MRCP is quite high and a mid-posterior insertion is the most common variant type. Insertion variations of CD, the low medial insertion in particular, are less common in patients with choledocholithiasis than controls.

Performance comparison: exome-sequencing as a single test replacing Sanger-sequencing

ABSTRACTSystematic performance comparing the results of exome-sequencing as a single test replacing Sanger-sequencing of targeted gene(s) is still lacking. In this study we compared Sanger-sequencing results of 258 genes to those obtained from next generation sequencing (NGS) using two exome-sequencing enrichment kits: Agilent-SureSelectQXT and Illumina-Nextera. Sequencing was performed on leukocytes and buccal-derived DNA from a single individual, and all 258 genes were sequenced a total of 11 times (using different sequencing methods and DNA sources). Sanger-sequencing was completed for all exons, including flanking ±8bp regions. For the 258 genes, NGS mean coverage was >20x for >98% and >91% of the regions targeted by SureSelect and Nextera, respectively. Overall, 449 variants were identified in at least one experiment, and 407/449 (90.6%) were detected by all. Of the 42 discordant variants, 23 were determined as true calls, summing-up to a truth set of 430 variants. Sensitivity of true-variant detection was 99% for Sanger-sequencing and 97%-100% for the NGS experiments. Mean false-positive rates were 3.7E-6 for Sanger-sequencing, 2.5E-6 for SureSelect-NGS and 5.2E-6 for Nextera-NGS. Our findings suggest a high overall concordance between Sanger-sequencing and NGS. Both methods demonstrated false positive and false negative calls and similar performances. Consequently, high clinical suspicion for a specific diagnosis should override negative results of either Sanger-sequencing or NGS.

Discrete regimes of information reception in non-antagonistic repeated game

The gain functions depend on the choices of players and time. The set of choices of the second player is changed in time according to one of some variants. The true variant is ascertained during the game. The current information about the set of choices and about partner's choices is received as sample data. An optimal discrete procedure of obtaining information is found that allows preserving the equilibrium.

Arnav: Site specific error models to identify variants in RNA

SummaryWe present Arnav (Analysis of RNA variants) a lightweight and easy-to-use statistical method for detecting mutations from RNA sequencing data. Site-specific error models allow Arnav to call variants with high specificity when the true variant allele fraction is unknown. We show the utility of Arnav by identifying variants using RNA sequencing data from the GTEx project.Availability and ImplementationArnav is implemented in C++ and is distributed under the GPL license at https://github.com/gatoravi/[email protected]

A comprehensive genomics solution for HIV surveillance and clinical monitoring in a global health setting

High-throughput viral genetic sequencing is needed to monitor the spread of drug resistance, direct optimal antiretroviral regimes, and to identify transmission dynamics in generalised HIV epidemics. Public health efforts to sequence HIV genomes at scale face three major technical challenges: (i) minimising assay cost and protocol complexity, (ii) maximising sensitivity, and (iii) recovering accurate and unbiased sequences of both the genome consensus and the within-host viral diversity. Here we present a novel, high-throughput, virus-enriched sequencing method and computational pipeline tailored specifically to HIV (veSEQ-HIV), which addresses all three technical challenges, and can be used directly on leftover blood drawn for routine CD4 testing. We demonstrate its performance on 1,620 plasma samples collected from consenting individuals attending 10 large urban clinics in Zambia, partners of HPTN 071 (PopART). We show that veSEQ-HIV consistently recovers complete HIV genomes from the majority of samples of different subtypes, and is also quantitative: the number of HIV reads per sample obtained by veSEQ-HIV estimates viral load without the need for additional testing. Both quantitativity and sensitivity were assessed on a subset of 126 samples with clinically measured viral loads, and with standardized quantification controls (VL 100 – 5,000,000 RNA copies/ml). Complete HIV genomes were recovered from 93% (85/91) of samples when viral load was over 1,000 copies per ml. The quantitative nature of the assay implies that variant frequencies estimated with veSEQ-HIV are representative of true variant frequencies in the sample. Detection of minority variants can be exploited for epidemiological analysis of transmission and drug resistance, and we show how the information contained in individual reads of a veSEQ-HIV sample can be used to detect linkage between multiple mutations associated with resistance to antiretroviral therapy. Less than 2% of reads obtained by veSEQ-HIV were identified as in silico contamination events using updates to the phyloscanner software (phyloscanner clean) that we show to be 95% sensitive and 99% specific at ‘decontaminating’ NGS data. The cost of the assay — approximately 45 USD per sample — compares favourably with existing VL and HIV genotyping tests, and provides the additional value of viral load quantification and inference of drug resistance with a single test. veSEQ-HIV is well suited to large public health efforts and is being applied to all ∼9000 samples collected for the HPTN 071-2 (PopART Phylogenetics) study.

Doubts and Faith

Three very different first-century Jewish authors hint at a possible deviation from the regime of anatomical testing of virginity as established by Deuteronomy 22. Both Josephus and Philo, in their paraphrases of the bloody-sheets pericope, strikingly leave out any mention of any physical remainder of the sexual act, thus deviating from the explicit model of Deuteronomy. In the end, however, Josephus, seems unlikely to be a true variant, likely avoiding rather than replacing the Deuteronomic standard. Philo, however, may well express a concern for spiritual, rather than (or in addition to) physical virginity. The contrast with Deuteronomy is even more pronounced in the Gospel of Matthew, where faith-based testing comes to replace physical testing.

An Explanation for the Reading ἐν µέσῳ αὐτῆς in LXX-Jer 29:14 in Light of Dalet-Resh Interchange

This article argues that in LXX-Jer 29, the rendering ἐν µέσῳ αὐτῆς “in her midst” suggests that the Greek translator had before him a true variant which read בצדה rather than בצרה, which the MT reads. The variant בצדה is supported by several other Greek versions. Furthermore, dalet-resh interchange is a common phenomenon exhibited by LXX-Jer 29 and the parallel Hebrew texts.

A second case of Marburg’s variant of multiple sclerosis with vasculitis and extensive demyelination

Marburg’s variant of multiple sclerosis is a rapidly progressive and malignant form of multiple sclerosis (MS) that usually leads to severe disability or death within weeks to months without remission. Few cases have been described in the literature since the original description by Marburg. The classic pathological findings usually include highly destructive zones of extensive demyelination, necrosis with dense cellular infiltrate, and giant reactive astrocytes. We report a case of a 31-year-old woman with Marburg’s variant of MS who, over a period of eight months, became totally disabled, blind, and quadriplegic, with vocal cord paralysis, requiring a tracheostomy. The patient underwent diagnostic stereotactic brain biopsy. Clinical findings, magnetic resonance imaging (MRI), serologic and cerebrospinal fluid (CSF) findings, and neuropathology are discussed. MRI showed extensive white matter involvement in the brain and spinal cord that continuously progressed over time. A diagnostic stereotactic brain biopsy revealed extensive active demyelination with unexpected finding of active vasculitis and fibrinoid necrosis with a vascular inflammatory cell infiltrate, including polymorphonuclear neutrophils and rare eosinophils. Serologic work-up for vasculitis and neuromyelitis optica was unremarkable and the CSF showed only one oligoclonal band (OCB) not present in serum. This is the second case of Marburg’s variant of MS that demonstrated both demyelination and vasculitis. In our case these features were demonstrated simultaneously, even though the demyelination was the predominant pathological finding. Since vasculitis is not a feature of classic MS, these findings pose the question as to whether Marburg’s variant of MS is a true variant or different entity altogether.