Comparison of different sequencing techniques with multiplex real-time PCR for detection to identify SARS-CoV-2 variants of concern

As different SARS-CoV-2 variants emerge and with the continuous evolution of sub-lineages of the delta and other variants, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, as many resource-poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies’ (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using the Illumina generated <15% ambiguous bases. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. Sub-consensus single nucleotide variations (SNV) were highly correlated between both platforms (R2=0.79) while indels showed a weaker correlation (R2=0.13).Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number of reads, generated less ambiguous bases and was significantly cheaper than Illumina sequencing technology.

Yes, we can use it: A formal test on the accuracy of low-pass nanopore long-read sequencing for mitophylogenomics + barcoding research using the Caribbean spiny lobster Panulirus argus

Whole mitogenomes or short fragments (e.g., 300-700 bp of the cox1 gene) are markers of choice for revealing within- and among-species genealogies. Protocols for sequencing and assembling mitogenomes include 'primer walking' or 'long PCR' followed by Sanger sequencing or low-coverage whole genome (LCWGS) sequencing with or without prior mitochondrial enrichment and Illumina sequencing. The aforementioned strategies assemble complete and accurate mitochondrial genomes but are time consuming and/or expensive. In this study, I tested first if mitogenomes can be sequenced from long-read nanopore sequencing data exclusively. Second, I explored the accuracy of the long-read assembled genomes by comparing them to a 'gold' standard reference mitogenome retrieved from the same individualusing Illumina sequencing. LC-WGS using a MinION ONT device and various de-novo and reference-based assembly pipelines did retrieve a complete and highly accurate mitogenome for the Caribbean spiny lobster Panulirus argus. Discordance between each of the long-read assemblies and the reference mitogenome was mostly due to indels at the flanks of homopolymer regions. Although not 'perfect', phylogenetic analyses using entire mitogenomes or a fragment of the cox1 gene demonstrated that mitogenomes assembled using long reads can reliably identify the sequenced specimen as belonging to P. argus and distinguish it from other closely and distantly related species in the same genus, family, and superorder. This study serves as a proof-of-concept for the future implementation of in-situ surveillance protocols using the MinION so to detect mislabeling in P. argus across its supply chain. Mislabeling detection will improve fishery management in this overexploited lobster. This study will additionally aid in decreasing costs for exploring meta-population connectivity in the Caribbean spiny lobster and will aid with the transfer of genomics technology to low-income and developing countries.

Draft Whole-Genome Sequence of Sphingobium sp. Strain PNB, a Versatile Polycyclic Aromatic Hydrocarbon-Degrading Bacterium

Sphingobium sp. strain PNB can completely degrade phenanthrene, naphthalene, and biphenyl as the sole carbon and energy source. The strain is also capable of cometabolizing benzo[a]pyrene, pyrene, acenaphthene, fluoranthene, etc. Here, we report the 5.69-Mb assembly and annotation of the genome sequence of strain PNB, obtained using Illumina sequencing.

Potato leafroll virus reduces Buchnera aphidocola titer and alters vector transcriptome responses

Viruses in the Luteoviridae family, such as Potato leafroll virus (PLRV), are transmitted by aphids in a circulative and nonpropagative mode. This means the virions enter the aphid body through the gut when they feed from infected plants and then the virions circulate through the hemolymph to enter the salivary glands before being released into the saliva. Although these viruses do not replicate in their insect vectors, previous studies have demonstrated viruliferous aphid behavior is altered and the obligate symbiont of aphids, Buchnera aphidocola, may be involved in transmission. Here we provide the transcriptome of green peach aphids (Myzus persicae) carrying PLRV and virus-free control aphids using Illumina sequencing. Over 150 million paired-end reads were obtained through Illumina sequencing, with an average of 19 million reads per library. The comparative analysis identified 134 differentially expressed genes (DEGs) between the M. persicae transcriptomes, including 64 and 70 genes that were up- and down-regulated in aphids carrying PLRV, respectively. Using functional classification in the GO databases, 80 of the DEGs were assigned to 391 functional subcategories at category level 2. The most highly up-regulated genes in aphids carrying PLRV were cytochrome p450s, genes related to cuticle production, and genes related to development, while genes related to heat shock proteins, histones, and histone modification were the most down-regulated. PLRV aphids had reduced Buchnera titer and lower abundance of several Buchnera transcripts related to stress responses and metabolism. These results suggest carrying PLRV may reduce both aphid and Buchnera genes in response to stress. This work provides valuable basis for further investigation into the complicated mechanisms of circulative and nonpropagative transmission.

Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation

We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.

Ceftazidime/avibactam resistance is associated with different mechanisms in KPC-producing Klebsiella pneumoniae strains

This study focused on Klebsiella pneumoniae isolates that were resistant or had low susceptibility to a combination of ceftazidime/avibactam. We aimed to investigate the mechanisms underlying this resistance. A total of 24 multi-drug resistant isolates of K. pneumoniae were included in the study. The phenotypic determination of carbapenemase presence was based on the CARBA NP test. NG-Test CARBA 5 was also performed, and it showed KPC production in 22 out 24 strains. The molecular characterisation of blaKPC carbapenemase gene, ESBL genes (blaCTX-M, blaTEM, and blaSHV) and porin genes ompK35/36 was performed using the PCR. Finally, ILLUMINA sequencing was performed to determine the presence of genetic mutations.Various types of mutations in the KPC sequence, leading to ceftazidime/avibactam resistance, were detected in the analysed resistant strains. We observed that KPC-31 harboured the D179Y mutation, the deletion of the amino acids 167–168, and the mutation of T243M associated with ceftazidime/avibactam resistance. The isolates that did not present carbapenemase alterations were found to have other mechanisms such as mutations in the porins. The mutations both on the KPC-3 enzyme and in the porins confirmed, that diverse mechanisms confer resistance to ceftazidime/avibactam in K. pneumoniae.

Predation of the Japanese keelback (Hebius vibakari Boie, 1826) by the Slender racer (Orientocoluber spinalis Peters, 1866)

Background The Slender racer (Orientocoluber spinalis Peters, 1866) has recently been reclassified to the new genus Orientocoluber from Hierophis. Ecological knowledge of this species is limited due to its highly mobile behavior. On 17 July 2020, we captured a female O. spinalis on Oeyeon Island, Boryeong-si, Republic of Korea, and collected its feces for a diet analysis. We observed snake scales from the collected feces and subsequently determined the prey species through morphological and molecular methods. Results We initially hypothesized that the extracted fecal sample scales belonged to H. vibakari, due to their thin keel and rhombus shape. We also amplified H. vibakari DNA from the extracted fecal sample using Illumina sequencing methods. Our morphological and molecular results suggest that O. spinalis predates H. vibakari on Oeyeon Island. Conclusion This is the first report of O. spinalis predating another snake species, ophiophagy, and implies that H. vibakari may be a crucial prey item for O. spinalis on Oeyeon Island.

BPHL SARS-CoV-2 Tiled Amplicon Illumina Sequencing v2

This protocol details the Florida Department of Health's Bureau of Public Health Laboratories' (BPHL) wet lab portion of our SARS-CoV-2 next generation sequencing workflow. The method is a tiled amplicon approach using ARTIC V3 primers. The amplicon generation was adapted from the Matteson protocol1. The library preparation is Illumina NexteraXT. Library pooling and normalization were adapted from the Gohl protocol3. This protocol is for loading a MiSeq, but we have had equal success running on iSeqs and NextSeqs as well. Up to 96 libraries can be run on a MiSeq and up to 384 on a NextSeq.

Strategy and Performance Evaluation of Low-Frequency Variant Calling for SARS-CoV-2 Using Targeted Deep Illumina Sequencing

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, constitutes a tremendous global health issue. Continuous monitoring of the virus has become a cornerstone to make rational decisions on implementing societal and sanitary measures to curtail the virus spread. Additionally, emerging SARS-CoV-2 variants have increased the need for genomic surveillance to detect particular strains because of their potentially increased transmissibility, pathogenicity and immune escape. Targeted SARS-CoV-2 sequencing of diagnostic and wastewater samples has been explored as an epidemiological surveillance method for the competent authorities. Currently, only the consensus genome sequence of the most abundant strain is taken into consideration for analysis, but multiple variant strains are now circulating in the population. Consequently, in diagnostic samples, potential co-infection(s) by several different variants can occur or quasispecies can develop during an infection in an individual. In wastewater samples, multiple variant strains will often be simultaneously present. Currently, quality criteria are mainly available for constructing the consensus genome sequence, and some guidelines exist for the detection of co-infections and quasispecies in diagnostic samples. The performance of detection and quantification of low-frequency variants using whole genome sequencing (WGS) of SARS-CoV-2 remains largely unknown. Here, we evaluated the detection and quantification of mutations present at low abundances using the mutations defining the SARS-CoV-2 lineage B.1.1.7 (alpha variant) as a case study. Real sequencing data were in silico modified by introducing mutations of interest into raw wild-type sequencing data, or by mixing wild-type and mutant raw sequencing data, to construct mixed samples subjected to WGS using a tiling amplicon-based targeted metagenomics approach and Illumina sequencing. As anticipated, higher variation and lower sensitivity were observed at lower coverages and allelic frequencies. We found that detection of all low-frequency variants at an abundance of 10, 5, 3, and 1%, requires at least a sequencing coverage of 250, 500, 1500, and 10,000×, respectively. Although increasing variability of estimated allelic frequencies at decreasing coverages and lower allelic frequencies was observed, its impact on reliable quantification was limited. This study provides a highly sensitive low-frequency variant detection approach, which is publicly available at https://galaxy.sciensano.be, and specific recommendations for minimum sequencing coverages to detect clade-defining mutations at certain allelic frequencies. This approach will be useful to detect and quantify low-frequency variants in both diagnostic (e.g., co-infections and quasispecies) and wastewater [e.g., multiple variants of concern (VOCs)] samples.