Biology subjects

Ramachandran, A.

Publications and source records attributed to Ramachandran, A..

27 records · Page 2Linked to original sources

bioRxiv2021

A metagenomic-based stufy of two sites from the Barbadian reef system

We study the microbiome of sea water collected from two locations of the Barbadian coral reefs. The two sites differ in several environmental and ecological variables including their endogenous benthic community in addition to their proximity to urban development and runoffs from inland watersheds. The composition of the microbial community was estimated using whole genome DNA shotgun sequencing. Although both sites exhibit a similar degree of richness, the less urbanized site (Maycocks reef at Hangmans Bay) is strongly concentrated with phototrophs. In comparison, the more urbanized location (Bellairs Research Institute) is enriched for copiotrophs, macroalgal symbionts and marine-related disease-bearing organisms from taxa scattered across the tree of life. Overall, our samples and associated measurements of chemical and environmental qualities of the water are in line with previous marine microbiome profiles of warm ocean surface waters. This suggests our approach captures salient information regarding the state of each coral reef site and sets the stage for larger longitudinal studies of coral reef dynamics in Barbados.

microbiology↗

bioRxiv2020

Electric-field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In a basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex is activated when it highly specifically binds to target DNA, and the activated complex non-specifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We recently discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab sample. We here combine this ITP purification with loop-mediated isothermal amplification, and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in 30 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays. Significance statementRapid, early-stage screening is especially crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR-diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps--factors which prolong the process and impede use in low resource settings. We here combine on-chip electric-field control in combination with CRIPSR biology to directly address these limitations of current CRISPR-diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes 30 min from raw sample to result, a significant improvement over existing diagnostic methods for COVID-19.

bioengineering↗

bioRxiv2020

HELLO: A hybrid variant calling approach

Next Generation Sequencing (NGS) technologies that cost-effectively characterize genomic regions and identify sequence variations using short reads are the current standard for genome sequencing. However, calling small indels in low-complexity regions of the genome using NGS is challenging. Recent advances in Third Generation Sequencing (TGS) provide long reads, which call large-structural variants accurately. However, these reads have context-dependent indel errors in low-complexity regions, resulting in lower accuracy of small indel calls compared to NGS reads. When both small and large-structural variants need to be called, both NGS and TGS reads may be available. Integration of the two data types with unique error profiles could improve robustness of small variant calling in challenging cases. However, there isnt currently such a method integrating both types of data. We present a novel method that integrates NGS and TGS reads to call small variants. We leverage the Mixture of Experts paradigm which uses an ensemble of Deep Neural Networks (DNN), each processing a different data type to make predictions. We present improvements in our DNN design compared to previous work such as sequence processing using one-dimensional convolutions instead of image processing using two-dimensional convolutions and an algorithm to efficiently process sites with many variant candidates, which help us reduce computations. Using our method to integrate Illumina and PacBio reads, we find a reduction in the number of erroneous small variant calls of up to ~30%, compared to the state-of-the-art using only Illumina data. We also find improvements in calling small indels in low-complexity regions.

bioinformatics↗

bioRxiv2019

High accuracy DNA sequencing on a small, scalable platform via electrical detection of single base incorporations

High throughput DNA sequencing technologies have undergone tremendous development over the past decade. Although optical detection-based sequencing has constituted the majority of data output, it requires a large capital investment and aggregation of samples to achieve optimal cost per sample. We have developed a novel electronic detection-based platform capable of accurately detecting single base incorporations. The GenapSys technology with its electronic detection modality allows the system to be compact, accessible, and affordable. We demonstrate the performance of the system by sequencing several different microbial genomes with varying GC content. The platform is capable of generating up to 2 Gb of high-quality nucleic acid sequence in a single run. We routinely generate sequence data that exceeds 99% raw accuracy with read lengths of up to 175 bp. Average quality scores remain above Q30 (99.9% raw sequencing accuracy) beyond 150 bp, with more than 85% of total bases at or above Q30. The utility of the platform is highlighted by targeted sequencing of the human genome. We show high concordance of SNP detection on the human NA12878 HapMap cell line with data generated on the Illumina sequencing platform. In addition, we sequenced a targeted panel of cancer-associated genes in a well characterized reference standard. With multiple library preparation approaches on this sample, we were able to identify low frequency mutations at expected allele frequencies.

genomics↗

bioRxiv2019

Diversity, biogeography, and evidence for endemism of SAR11 bacteria from the Arctic Ocean

The Arctic Ocean is relatively isolated from other oceans and consists of strongly stratified water masses with distinct histories, nutrient, temperature and salinity characteristics, therefore providing an optimal environment to investigate local adaptation. The globally distributed SAR11 bacterial group consists of multiple ecotypes that are associated with particular marine environments, yet relatively little is known about Arctic SAR11 diversity. Here, we examined SAR11 diversity using ITS analysis and metagenome-assembled genomes (MAGs). Arctic SAR11 assemblages were comprised of the S1a, S1b, S2, and S3 clades, and structured by water mass and depth. The fresher surface layer was dominated by an ecotype (S3-derived P3.2) previously associated with Arctic and brackish water. In contrast, deeper waters of Pacific origin were dominated by the P2.3 ecotype of the S2 clade, within which we identified a novel subdivision (P2.3s1) that was rare outside the Arctic Ocean. Arctic S2-derived SAR11 MAGs were restricted to high latitudes and included MAGs related to the recently defined S2b subclade, a finding consistent with bi-polar ecotypes and Arctic endemism. These results place the stratified Arctic Ocean into the SAR11 global biogeography and have identified SAR11 lineages for future investigation of adaptive evolution in the Arctic Ocean.

microbiology↗

bioRxiv2018

Genomic Surveillance for Antimicrobial Resistance in Mannheimia haemolytica Using Nanopore Single Molecule Sequencing Technology

Disruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain of Mannheimia haemolytica, isolated from pneumonic bovine lung samples, were respectively sequenced at 146x and 111x coverage with Oxford Nanopore Technologies MinION. De novo assembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including beta lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Antibiotic resistance phenotypes of the M. haemolytica strains were confirmed with minimum inhibitory concentration (MIC) assays. The sequencing capacity of highly portable MinION devices was verified by sub-sampling sequencing reads; potential for antimicrobial resistance determined by identification of resistance genes in the draft assemblies with as little as 5,437 MinION reads corresponded to all classes of MIC assays. The resulting quality assemblies and AMR gene annotation highlight efficiency of ultra long-read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.

microbiology↗

bioRxiv2018

Mitochondrial RNA capping: highly efficient 5’-RNA capping with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

biochemistry↗

bioRxiv2018

Microbial life under ice: metagenome diversity and in situ activity of Verrucomicrobia in seasonally ice-covered lakes

Northern lakes are ice-covered for a large part of the year, yet our understanding of microbial diversity and activity during winter lags behind that of the ice-free period. In this study, we investigated under-ice diversity and metabolism of Verrucomicrobia in seasonally ice-covered lakes in temperate and boreal regions of Quebec, Canada using 16S rRNA sequencing, metagenomics and metatranscriptomics. Verrucomicrobia, particularly the V1, V3 and V4 subdivisions, were abundant during ice-covered periods. A diversity of Verrucomicrobia genomes were reconstructed from Quebec lake metagenomes. Several genomes were associated with the ice-covered period and were represented in winter metatranscriptomes, supporting the notion that Verrucomicrobia are metabolically active under ice. Verrucomicrobia transcriptome analysis revealed a range of metabolisms potentially occurring under ice, including carbohydrate degradation, glycolate utilization, scavenging of chlorophyll degradation products, and urea use. Genes for aerobic sulfur and hydrogen oxidation were expressed, suggesting chemolithotrophy may be an adaptation to conditions where labile carbon may be limited. The expression of genes for flagella biosynthesis and chemotaxis was detected, suggesting Verrucomicrobia may be actively sensing and responding to winter nutrient pulses, such as phytoplankton blooms. These results increase our understanding on the diversity and metabolic processes occurring under ice in northern lakes ecosystems.

microbiology↗

bioRxiv2017

Whole genome sequencing-based detection of antimicrobial resistance and virulence in non-typhoidal Salmonella enterica isolated from wildlife

The aim of this study was to generate a reference set of Salmonella enterica genomes isolated from wildlife from the United States and to determine the antimicrobial resistance and virulence gene profile of the isolates from the genome sequence data. We sequenced the whole genomes of 103 Salmonella isolates sampled between 1988 and 2003 from wildlife and exotic pet cases that were submitted to the Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, Oklahoma. Among 103 isolates, 50.48% were from wild birds, 0.9% was from fish, 24.27% each were from reptiles and mammals. 50.48% isolates showed resistance to at least one antibiotic. Resistance against the aminoglycoside streptomycin was most common while 9 isolates were found to be multi-drug resistant having resistance against more than three antibiotics. Determination of virulence gene profile revealed that the genes belonging to csg operons, the fim genes that encode for type 1 fimbriae and the genes belonging to type III secretion system were predominant among the isolates. The universal presence of fimbrial genes and the genes encoded by pathogenicity islands 1-2 among the isolates we report here indicates that these isolates could potentially cause disease in humans. Therefore, the genomes we report here could be a valuable reference point for future traceback investigations when wildlife is considered to be the potential source of human Salmonellosis.

microbiology↗