Multiplex Sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13751-13965 and 23941-24106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64-Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13730 and 23929, whereas the other isolates possess T at these positions. The genetic variations observed suggest that the naturally occurring genome variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel. The possible impact of other genetic sequence variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel, hence, should be considered.

Whole-Genome Sequencing and Phylogenetic Analysis of Francisella tularensis Vaccine Strain 15 NIIEG

Objective of the study was to conduct whole-genome sequencing of the vaccine strain Francisella tularensis 15 NIIEG and determine, based on the results, its phylogenetic relationships and the genetic organization features.Materials and methods. Whole-genome sequencing of F. tularensis 15 NIIEG strain was performed on Ion PGM (Ion Torrent, USA) and MinIon (Oxford Nanopore Technologies, UK) platforms. Alignment of readings obtained to the whole-genome of F. tularensis subsp. holarctica LVS (CP009694, USA, 2015) was performed using the software package DNASTAR Lasergene 15.3. Hybrid assembly of reads into contigs was performed by means of Unicycler v. 0.4.4, using data obtained by semiconductor sequencing technology (Ion PGM) and nanopore sequencing (MinIon). Phylogenetic analysis was performed on the basis of single nucleotide polymorphism (SNPs) data located in the core part of F. tularensis genome. Maximum parsimony algorithm was used to construct a dendrogram using the obtained data of common SNP-matrix.Results and discussion. The close relations of F. tularensis 15 NIIEG strain with F. tularensis LVS vaccine strain used in the countries of Western Europe and North America was confirmed. Searching for common single mutations characteristic of F. tularensis 15 vaccine strains of NIIEG and LVS, permitted to find 5 unique SNPs that distinguish them from all other 228 F. tularensis strains used in the comparison. Comparative genomic analysis ofF. tularensis 15 NIIEG vaccine strain and virulent strains revealed in its structure two extensive 526 bp deletions (genes pilA and pilE) and 1480 bp (genes encoding lipoprotein). Similar deletions are also present in the genome of the F. tularensis LVS vaccine strain.

Phylogenetic Analysis of Yersinia pestis Strains of Medieval Biovar, Isolated in Precaspian North-Western Steppe Plague Focus in the XX Century

Objective of the study – comparative phylogenetic analysis of Yersinia pestis strains, isolated in Precaspian North-Western steppe focus in 1924–1926, 1972, and 1986–1990 to understand the causes of focal reactivation during different time periods of the XX century.Materials and methods. The work included 30 strains of Yersinia pestis from Precaspian North-Western steppe natural focus and adjacent plague foci. Whole genome sequencing of eight Y. pestis strains from the former was carried out. Also whole-genome sequences of 16 strains from neighboring natural foci were used. Whole-genome sequencing of Y. pestis strains was conducted in Ion PGM system (Life technologies). SNPs search across the core genome was performed using software package Wombac 2.0. Tree diagram Maximum Likelihood, HKU85 model, was constructed to analyze phylogenetic relations.Results and discussion. It is established that in early XX century (1924–1926), strains of phylogenetic branches 2.MED4 and 2.MED1, belonging to medieval biovar, main subspecies, circulated on Ergenin Upland in the Precaspian North-Western steppe natural focus. Later on they became extinct in the territory. It is shown that the strains, isolated on Ergenin Upland in 1972, constituted a common subcluster on the dendrogram with the strains from low-mountain and piedmont plague foci of Caucasus and Transcaucasia, dated the same time period. It was inferred that epizootic manifestations on Ergenin upland in 1972, after a long recess since 1938, were caused by importation of Y. pestis strains from low-mountain natural plague foci of Caucasus and Transcaucasia. It was noted that expansion of Caucasian strains was of short-term character, and plague infected animals have not been found on Ergenin Upland since 1974 (including modern period). It is established that Y. pestis strains isolated in the eastern part of Precaspian North-Western steppe focus between 1986 and 1990, do not have close genetic relation to the strains that circulated on Ergenin Upland in 1924–1926 and 1972. It is determined that each epizootic period (1913–1938 and 1972–1973) in Precaspian North-Western steppe natural focus culminated in the elimination of the circulating Y. pestis strains and rehabilitation of the focal territory.

Evaluating metabarcoding to analyse diet composition of species foraging in anthropogenic landscapes using Ion Torrent and Illumina sequencing

DNA metabarcoding of faecal samples is being successfully used to study the foraging niche of species. We assessed the ability of two benchtop high-throughput sequencing (HTS) platforms, to identify a large taxonomic array of food items from domestic cats Felis silvestris catus, including prey and human-related food taxa (pet food and leftovers leaving undetectable solid remains in faeces). Scats from a captive feeding trial (n=41) and from free-ranging individuals (n=326) were collected and analysed using a cytb mini-barcode in independent PCR duplicates on the Ion PGM and the MiSeq platforms. Outputs from MiSeq were more sensitive and reproducible than those from Ion PGM due to a higher sequencing depth and sequence quality on MiSeq. DNA from intact prey taxa was detected more often (82% of the expected occurrences) than DNA from pet food (54%) and raw fish and meat (31%). We assumed that this variability was linked to different degree of DNA degradation: The Ion PGM detected significantly less human-linked food, birds, field voles, murids and shrews in the field-collected samples than the MiSeq platform. Pooling the replicates from both platforms and filtering the data allowed identification of at least one food item in 87.4% of the field-collected samples. Our DNA metabarcoding approach identified 29 prey taxa, of which 25 to species level (90% of items) including 9 rodents, 3 insectivores, 12 birds and 1 reptile and 33 human-related food taxa of which 23 were identified to genus level (75% of items). Our results demonstrate that using HTS platforms such as MiSeq, which provide reads of sufficiently high quantity and quality, with sufficient numbers of technical replicates, is a robust and non-invasive approach for further dietary studies on animals foraging on a wide range of food items in anthropogenic landscapes.