Assembly-free rapid differential gene expression analysis in non-model organisms using DNA-protein alignment.

BACKGROUND: RNA-seq is being increasingly adopted for gene expression studies in a panoply of non-model organisms, with applications spanning the fields of agriculture, aquaculture, ecology, and environment. For organisms that lack a well-annotated reference genome or transcriptome, a conventional RNA-seq data analysis workflow requires constructing a de-novo transcriptome assembly and annotating it against a high-confidence protein database. The assembly serves as a reference for read mapping, and the annotation is necessary for functional analysis of genes found to be differentially expressed. However, assembly is computationally expensive. It is also prone to errors that impact expression analysis, especially since sequencing depth is typically much lower for expression studies than for transcript discovery. RESULTS: We propose a shortcut, in which we obtain counts for differential expression analysis by directly aligning RNA-seq reads to the high-confidence proteome that would have been otherwise used for annotation. By avoiding assembly, we drastically cut down computational costs - the running time on a typical dataset improves from the order of tens of hours to under half an hour, and the memory requirement is reduced from the order of tens of Gbytes to tens of Mbytes. We show through experiments on simulated and real data that our pipeline not only reduces computational costs, but has higher sensitivity and precision than a typical assembly-based pipeline. A Snakemake implementation of our workflow is available at: https://bitbucket.org/project_samar/samar . CONCLUSIONS: The flip side of RNA-seq becoming accessible to even modestly resourced labs has been that the time, labor, and infrastructure cost of bioinformatics analysis has become a bottleneck. Assembly is one such resource-hungry process, and we show here that it can be avoided for quick and easy, yet more sensitive and precise, differential gene expression analysis in non-model organisms.

Comparative transcriptome profiling of heat stress response of the mangrove crab Scylla serrata across sites of varying climate profiles.

BACKGROUND: The fishery and aquaculture of the widely distributed mangrove crab Scylla serrata is a steadily growing, high-value, global industry. Climate change poses a risk to this industry as temperature elevations are expected to threaten the mangrove crab habitat and the supply of mangrove crab juveniles from the wild. It is therefore important to understand the genomic and molecular basis of how mangrove crab populations from sites with different climate profiles respond to heat stress. Towards this, we performed RNA-seq on the gill tissue of S. serrata individuals sampled from 3 sites (Cagayan, Bicol, and Bataan) in the Philippines, under normal and heat-stressed conditions. To compare the transcriptome expression profiles, we designed a 2-factor generalized linear model containing interaction terms, which allowed us to simultaneously analyze within-site response to heat-stress and across-site differences in the response. RESULTS: We present the first ever transcriptome assembly of S. serrata obtained from a data set containing 66 Gbases of cleaned RNA-seq reads. With lowly-expressed and short contigs excluded, the assembly contains roughly 17,000 genes with an N50 length of 2,366 bp. Our assembly contains many almost full-length transcripts - 5229 shrimp and 3049 fruit fly proteins have alignments that cover >80% of their sequence lengths to a contig. Differential expression analysis found population-specific differences in heat-stress response. Within-site analysis of heat-stress response showed 177, 755, and 221 differentially expressed (DE) genes in the Cagayan, Bataan, and Bicol group, respectively. Across-site analysis showed that between Cagayan and Bataan, there were 389 genes associated with 48 signaling and stress-response pathways, for which there was an effect of site in the response to heat; and between Cagayan and Bicol, there were 101 such genes affecting 8 pathways. CONCLUSION: In light of previous work on climate profiling and on population genetics of marine species in the Philippines, our findings suggest that the variation in thermal response among populations might be derived from acclimatory plasticity due to pre-exposure to extreme temperature variations or from population structure shaped by connectivity which leads to adaptive genetic differences among populations.

Jointly aligning a group of DNA reads improves accuracy of identifying large deletions.

Performing sequence alignment to identify structural variants, such as large deletions, from genome sequencing data is a fundamental task, but current methods are far from perfect. The current practice is to independently align each DNA read to a reference genome. We show that the propensity of genomic rearrangements to accumulate in repeat-rich regions imposes severe ambiguities in these alignments, and consequently on the variant calls-with current read lengths, this affects more than one third of known large deletions in the C. Venter genome. We present a method to jointly align reads to a genome, whereby alignment ambiguity of one read can be disambiguated by other reads. We show this leads to a significant improvement in the accuracy of identifying large deletions (≥20 bases), while imposing minimal computational overhead and maintaining an overall running time that is at par with current tools. A software implementation is available as an open-source Python program called JRA at https://bitbucket.org/jointreadalignment/jra-src.

Combining probabilistic alignments with read pair information improves accuracy of split-alignments.

Motivation: Split-alignments provide base-pair-resolution evidence of genomic rearrangements. In practice, they are found by first computing high-scoring local alignments, parts of which are then combined into a split-alignment. This approach is challenging when aligning a short read to a large and repetitive reference, as it tends to produce many spurious local alignments leading to ambiguities in identifying the correct split-alignment. This problem is further exacerbated by the fact that rearrangements tend to occur in repeat-rich regions. Results: We propose a split-alignment technique that combats the issue of ambiguous alignments by combining information from probabilistic alignment with positional information from paired-end reads. We demonstrate that our method finds accurate split-alignments, and that this translates into improved performance of variant-calling tools that rely on split-alignments. Availability and implementation: An open-source implementation is freely available at: https://bitbucket.org/splitpairedend/last-split-pe. Supplementary information: Supplementary data are available at Bioinformatics online.

A bioinformatician's guide to the forefront of suffix array construction algorithms.

The suffix array and its variants are text-indexing data structures that have become indispensable in the field of bioinformatics. With the uninitiated in mind, we provide an accessible exposition of the SA-IS algorithm, which is the state of the art in suffix array construction. We also describe DisLex, a technique that allows standard suffix array construction algorithms to create modified suffix arrays designed to enable a simple form of inexact matching needed to support 'spaced seeds' and 'subset seeds' used in many biological applications.

An approximate Bayesian approach for mapping paired-end DNA reads to a reference genome.

SUMMARY: Many high-throughput sequencing experiments produce paired DNA reads. Paired-end DNA reads provide extra positional information that is useful in reliable mapping of short reads to a reference genome, as well as in downstream analyses of structural variations. Given the importance of paired-end alignments, it is surprising that there have been no previous publications focusing on this topic. In this article, we present a new probabilistic framework to predict the alignment of paired-end reads to a reference genome. Using both simulated and real data, we compare the performance of our method with six other read-mapping tools that provide a paired-end option. We show that our method provides a good combination of accuracy, error rate and computation time, especially in more challenging and practical cases, such as when the reference genome is incomplete or unavailable for the sample, or when there are large variations between the reference genome and the source of the reads. An open-source implementation of our method is available as part of Last, a multi-purpose alignment program freely available at http://last.cbrc.jp. CONTACT: martin@cbrc.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RicePilaf: a post-GWAS/QTL dashboard to integrate pangenomic, coexpression, regulatory, epigenomic, ontology, pathway, and text-mining information to provide functional insights into rice QTLs and GWAS loci.

BACKGROUND: As the number of genome-wide association study (GWAS) and quantitative trait locus (QTL) mappings in rice continues to grow, so does the already long list of genomic loci associated with important agronomic traits. Typically, loci implicated by GWAS/QTL analysis contain tens to hundreds to thousands of single-nucleotide polmorphisms (SNPs)/genes, not all of which are causal and many of which are in noncoding regions. Unraveling the biological mechanisms that tie the GWAS regions and QTLs to the trait of interest is challenging, especially since it requires collating functional genomics information about the loci from multiple, disparate data sources. RESULTS: We present RicePilaf, a web app for post-GWAS/QTL analysis, that performs a slew of novel bioinformatics analyses to cross-reference GWAS results and QTL mappings with a host of publicly available rice databases. In particular, it integrates (i) pangenomic information from high-quality genome builds of multiple rice varieties, (ii) coexpression information from genome-scale coexpression networks, (iii) ontology and pathway information, (iv) regulatory information from rice transcription factor databases, (v) epigenomic information from multiple high-throughput epigenetic experiments, and (vi) text-mining information extracted from scientific abstracts linking genes and traits. We demonstrate the utility of RicePilaf by applying it to analyze GWAS peaks of preharvest sprouting and genes underlying yield-under-drought QTLs. CONCLUSIONS: RicePilaf enables rice scientists and breeders to shed functional light on their GWAS regions and QTLs, and it provides them with a means to prioritize SNPs/genes for further experiments. The source code, a Docker image, and a demo version of RicePilaf are publicly available at https://github.com/bioinfodlsu/rice-pilaf.

Clinicodemographic profile of chronic liver disease patients at a tertiary care hospital: a retrospective analysis.

With the global burden of chronic liver disease (CLD) on the rise, especially due to the rise in obesity and metabolic syndrome, a third-world country like Nepal faces a different problem. With alcohol intake being rooted in Nepalese culture, alcoholic liver disease (ALD) is the most common cause of CLD in our society. Methods: This is a retrospective observational study conducted in the inpatient ward of the Department of Gastroenterology at the University in Nepal. Ethical approval was taken from the Institutional Review Committee, and a structured questionnaire format was used to record the data retrospectively using admission log books and admission sheets. Demographic data regarding age, sex, and address were collected, while the form of decompensation during presentation was used as a source of clinical data. For statistical analysis, see SPSS 21 (IBM Corp., Released 2012. IBM SPSS Statistics for Windows, Version 21.0; IBM Corp.) was used. Results: A male-to-female ratio of 2:1 was found, with ALD the most common cause of CLD in admitted patients. Similarly, the majority of patients were admitted due to ascites, which was compounded by spontaneous bacterial peritonitis. 93.60% of patients admitted with CLD had a deranged prothrombin time, while only about a third of patients had elevated aspartate aminotransferase and/or alanine aminotransferase. Conclusion: The large burden of ALD highlights the importance of awareness programs at the community level, which have been neglected till date.

Protein embeddings improve phage-host interaction prediction.

With the growing interest in using phages to combat antimicrobial resistance, computational methods for predicting phage-host interactions have been explored to help shortlist candidate phages. Most existing models consider entire proteomes and rely on manual feature engineering, which poses difficulty in selecting the most informative sequence properties to serve as input to the model. In this paper, we framed phage-host interaction prediction as a multiclass classification problem that takes as input the embeddings of a phage's receptor-binding proteins, which are known to be the key machinery for host recognition, and predicts the host genus. We explored different protein language models to automatically encode these protein sequences into dense embeddings without the need for additional alignment or structural information. We show that the use of embeddings of receptor-binding proteins presents improvements over handcrafted genomic and protein sequence features. The highest performance was obtained using the transformer-based protein language model ProtT5, resulting in a 3% to 4% increase in weighted F1 and recall scores across different prediction confidence thresholds, compared to using selected handcrafted sequence features.

Intramuscular hydatid cyst of paraspinal muscle: A diagnostic challenge.

Hydatid cyst is a zoonosis caused by ingestion of eggs of Echinococcus-a tapeworm. Following ingestion of the eggs, oncosphere larvae are released which penetrate the intestinal wall to invade various organs of the body including liver, lungs, and skeletal muscles via. blood vessels. Due to their contractile nature and high lactic acid content, skeletal muscles are typically not a suitable site for attachment of hydatid cyst. In our case report presented below, we report a rare case of an isolated hydatid cyst of paraspinal muscle presenting as a slow-growing asymptomatic mass in the back, posing a clinical diagnostic dilemma.

Superior vena cava syndrome in a child with mediastinal mass: A case report.

Facial puffiness as a consequence of superior vena cava syndrome (SVCS) can be a presentation from which the physician would have to conjecture a diagnosis of a mediastinal mass including lymphomas. Pediatric SVCS can rapidly progress and pose a greater challenge for airway protection as was in our case.

Prolonged labor presenting as vulvar edema during pregnancy.

Isolated vulvar edema is a rare complaint during pregnancy with a long list of differential diagnosis. Here, we describe a case of vulvar edema due to obstructed labor presenting to a rural primary healthcare center in Nepal.

Biliary Cripple and the Spectrum of Complications following Cholecystectomy: A Case Report.

Bile duct injury is a complication seen during cholecystectomy. Here, we highlight the occurrence of bile duct injury (BDI) during an open cholecystectomy who underwent hepaticojejunostomy (HJ), later presenting with a stricture of HJ. Percutaneous transhepatic biliary drainage (PTBD) was performed which led to the development of hepatic artery injury.

Mesenteric Ischemia and Its Need for Timely Recognition and Management.

Mesenteric ischemia is a fatal vascular emergency of the small intestine which, if not diagnosed and treated in time, has a very high mortality rate. Presenting with nonspecific symptoms such as abdominal pain, nausea, constipation, tachycardia, and gastrointestinal bleeds, it can masquerade as other causes of acute abdomen, particularly bowel obstruction. Ideal laboratory tests and markers are still lacking due to complexity in bowel's anatomy, physiology, blood supply, and drainage. We report 10 cases of mesenteric ischemia presented in our center with their demography, laboratory findings, approach to diagnosis, and treatment along with their outcomes at discharge. Out of the ten cases, six cases presented with signs and symptoms of acute bowel obstruction without passage of stool and one with passage of black stool. These seven patients underwent laparotomy, while the remaining three cases who either presented with milder symptoms or refused surgical interventions were managed conservatively. All patients were diagnosed with either acute or chronic mesenteric ischemia based on their operative and/or radiographic findings.

Hemoglobin J in a patient with severe anemia, a case report from Nepal.

Introduction: Hemoglobin J is defined by a faster movement towards anode when compared with the normal hemoglobin A. Though a pathologically distinct entity from the normal HbA, it remains clinically silent due to little physiological difference as exemplified by a similar oxygen binding capacity between the two. Though cases of symptomatic HbJ have been reported, it is uncommon. Hence, further explanations should be sought in such cases. Case presentation: Our case report exemplifies the presence of an alpha thalassemia trait along with HbJ in a symptomatic case of anemia from rural Nepal. Discussion: CE-HPLC complemented by electrophoresis, is the method of choice for characterizing various hemoglobin variants including Hb J. Hb J presents as elevated P3 peak on HPLC while thalassemia is detected by the presence of eluted proteins at the retention time between 0 and 1 minutes. P3 peak up to 6% is considered normal, values 6%-12% indicates suboptimal specimen and values greater than 15% indicates Hb J. Conclusion: Variants of hemoglobin including HbJ variant is detected using HPLC technique. Mostly clinically silent, if HbJ is associated with anemia, search for a concomitant cause should be sought one of them being alpha thalassemia when iron deficiency has been ruled out by a serum iron profile.

Duodenal gastrointestinal stromal tumor: A case report.

Introduction: Gastrointestinal tumor (GIT) is an uncommon gastrointestinal tumor most commonly arising in the stomach. Duodenum is an uncommon site accounting for only about 3-5% of cases. Case presentation: In this case report, we present a case of high-risk duodenal GIST and review its management strategies. Discussion: An abdominal mass and gastrointestinal (GI) bleeding are its usual presentation, however it may be lost among the long list of differentials of an abdominal mass and GI bleeding, if a high index of suspicion is not maintained. Surgery, with or without tyrosine kinase inhibitors like imatinib mesylate, has been the cornerstone in management of GIST. Conclusion: This case underlined the importance of duodenal GIST as a cause of GI bleed and abdominal mass as well as shone light upon historical developments, current updates and managements of GISTs.

Mesenteric inflammatory myofibroblastic tumor mimicking acute appendicitis: A case report.

Introduction: An Inflammatory myofibroblastic tumor, a neoplasm of intermediate biological potential, of the small bowel mesentery, is a rare tumor, most commonly reported in but not confined to the pediatric age group. Case presentation: This case report underlines a case of a (small bowel) mesentery IMT in an adult female presenting with recurrent symptoms similar to acute appendicitis. Discussion: It can present with symptoms similar to acute appendicitis necessitating a high index of suspicion for its prompt diagnosis. Treatment primarily includes surgical resection with recent advances in targeted therapy with tyrosine kinase inhibitors showing promising results. Conclusion: IMTs can present as clinical as well as histopathological mimickers of a variety of diseases especially in the abdomen. Prompt diagnosis requires both imaging and histopathological examination.

A Ruptured Pseudoaneurysm of an Anomalous Gastroduodenal Artery: A Rare Presentation.

Gastroduodenal artery (GDA) anomalies are a rare entity. Rupture of such anomalies can present with a diagnostic challenge. In this report, we describe a case of ruptured pseudoaneurysm of an anomalous GDA arising directly from the aorta presenting with recurrent abdominal pain and anemia. The diagnosis was made on computed tomography scan which showed acute retroperitoneal fluid collection. Further angiographic intervention highlighted the anomalous GDA arising directly from the aorta.

A Simplified Description of Child Tables for Sequence Similarity Search.

Finding related nucleotide or protein sequences is a fundamental, diverse, and incompletely-solved problem in bioinformatics. It is often tackled by seed-and-extend methods, which first find "seed" matches of diverse types, such as spaced seeds, subset seeds, or minimizers. Seeds are usually found using an index of the reference sequence(s), which stores seed positions in a suffix array or related data structure. A child table is a fundamental way to achieve fast lookup in an index, but previous descriptions have been overly complex. This paper aims to provide a more accessible description of child tables, and demonstrate their generality: they apply equally to all the above-mentioned seed types and more. We also show that child tables can be used without LCP (longest common prefix) tables, reducing the memory requirement.