CAIM: Coverage-based Analysis for Identification of Microbiome.

Accurate taxonomic profiling of microbial taxa in a metagenomic sample is vital to gain insights into microbial ecology. Recent advancements in sequencing technologies have contributed tremendously toward understanding these microbes at species resolution through a whole shotgun metagenomic (WMS) approach. In this study, we developed a new bioinformatics tool, CAIM, for accurate taxonomic classification and quantification within both long- and short-read metagenomic samples using an alignment-based method. CAIM depends on two different containment techniques to identify species in metagenomic samples using their genome coverage information to filter out false positives rather than the traditional approach of relative abundance. In addition, we propose a nucleotide-count based abundance estimation, which yield lesser root mean square error than the traditional read-count approach. We evaluated the performance of CAIM on 28 metagenomic mock communities and 2 synthetic datasets by comparing it with other top-performing tools. CAIM maintained a consitently good performance across datasets in identifying microbial taxa and in estimating relative abundances than other tools. CAIM was then applied to a real dataset sequenced on both Nanopore (with and without amplification) and Illumina sequencing platforms and found high similality of taxonomic profiles between the sequencing platforms. Lastly, CAIM was applied to fecal shotgun metagenomic datasets of 232 colorectal cancer patients and 229 controls obtained from 4 different countries and primary 44 liver cancer patients and 76 controls. The predictive performance of models using the genome-coverage cutoff was better than those using the relative-abundance cutoffs in discriminating colorectal cancer and primary liver cancer patients from healthy controls with a highly confident species markers. Key Points: Metagenomic coverage is an important index to obtain highly accurate species identification by reducing false positives from whole shotgun metagenomic data.Comparative analyses of CAIM and other bioinformatics tools for species identification on many mock community whole shotgun metagenomic datasets generated by short-read and long-read sequencing and synthetic datasets were performed, showing that CAIM has a very good performance compared with the other tools.Using the metagenomic coverage approach through CAIM improves the predictive power of species biomarkers identified from in stool samples of colorectal cancer and primary liver datasets.

Genomic surveillance of SARS-CoV-2 using long-range PCR primers.

Introduction: Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). Methods: In this study we used a set of seven long-range PCR primer pairs to sequence clinical isolates of SARS-CoV-2 on Oxford Nanopore sequencer. These long-range primers generate seven amplicons approximately 4500 bp that covered whole genome of SARS-CoV-2. One of these regions includes the full-length S-gene by using a set of flanking primers. We also evaluated the performance of these long-range primers with Midnight primers by sequencing 94 clinical isolates in a Nanopore flow cell. Results and discussion: Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias. The key finding of this study is that long range primers can be used in single-molecule sequencing of RNA viruses in surveillance of emerging variants. We also show that by designing primers flanking the S-gene, we can obtain reliable identification of SARS-CoV-2 variants.

Physiological and cognitive changes after treatments of cyclophosphamide, methotrexate, and fluorouracil: implications of the gut microbiome and depressive-like behavior.

Introduction: Chemotherapy-induced cognitive impairment colloquially referred to as chemobrain is a poorly understood phenomenon affecting a highly variable proportion of patients with breast cancer. Here we investigate the association between anxiety and despair-like behaviors in mice treated with cyclophosphamide, methotrexate, and fluorouracil (CMF) along with host histological, proteomic, gene expression, and gut microbial responses. Methods: Forced swim and sociability tests were used to evaluate depression and despair-like behaviors. The tandem mass tag (TMT) proteomics approach was used to assess changes in the neural protein network of the amygdala and hippocampus. The composition of gut microbiota was assessed through 16S rRNA gene sequencing. Finally, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate changes in intestinal gap junction markers. Results and discussion: We observed that CMF induced social and despair-like behavior in mice 96 hours following treatment. Proteomic analysis identified changes in various proteins related to progressive neurological disease, working memory deficit, primary anxiety disorder, and gene expression revealing increases in NMDA and AMPA receptors in both the hippocampus and the amygdala because of CMF treatment. These changes finally, we observed immediate changes in the microbial population after chemotherapy treatment, with a notable abundance of Muribaculaceae and Romboutsia which may contribute to changes seen in the gut.

Genomic Surveillance of SARS-CoV-2 Using Long-Range PCR Primers.

Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence the ~30,000 nucleotide SARS-CoV-2 genome that use a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). This study uses seven long-range PCR primers with an amplicon size of ~4500 bp to tile across the complete SARS-CoV-2 genome. One of these regions includes the full-length S-gene by using a set of flanking primers. Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias.

Severe Acute Respiratory Syndrome Coronavirus-2 Lambda Variant Collected from a Child from Arkansas and Sequenced.

An eleven-year-old tested positive for SARS-CoV-2 Lambda variant. Sequencing was performed on the Oxford Nanopore and the Illumina NextSeq 500. Both platforms identified all 7 of the synonymous mutations in the sample, while all 28 nonsynonymous mutations were identified from Oxford Nanopore and 20 nonsynonymous mutations were identified from Illumina.

16S rRNA Gene Amplicon Profiling of Baby and Adult Captive Elephants in Thailand.

Here, we present a 16S rRNA gene amplicon sequence data set and profiles demonstrating the bacterial diversity of baby and adult elephants from four different geographical locations in Thailand. The dominant phyla among baby and adult elephants were Bacteroidetes, Firmicutes, Proteobacteria, Kiritimatiellaeota, Euryarchaeota, and Tenericutes.

Rapid Sequencing of Multiple RNA Viruses in Their Native Form.

Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel. Mapping of the generated sequences on the reference genomes showed 100% length recovery with up to 97% identity. This work provides a proof of principle and the validity of this strategy, opening up a wide range of applications to study RNA viruses.