Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses.

BACKGROUND: Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols. METHODS: The genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood-saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results. RESULTS: The quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood-saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030-0.9998 for SNVs and between 0.8883-0.9991 for small-indels in the case of the WGS protocol, and between 0.8643-0.999 for SNVs and between 0.7781-1.000 for small-indels in the case of the WES protocol. CONCLUSION: Saliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used.

Distribution of SARS-CoV-2 Lineages in the Czech Republic, Analysis of Data from the First Year of the Pandemic.

In the Czech Republic, the current pandemic led to over 1.67 million SARS-CoV-2- positive cases since the recording of the first case on 1 March 2020. SARS-CoV-2 genome analysis is an important tool for effective real-time quantitative PCR (RT-qPCR) diagnostics, epidemiology monitoring, as well as vaccination strategy. To date, there is no comprehensive report on the distribution of SARS-CoV-2 genome variants in either the Czech Republic, including Central and Eastern Europe in general, during the first year of pandemic. In this study, we have analysed a representative cohort of SARS-CoV-2 genomes from 229 nasopharyngeal swabs of COVID-19 positive patients collected between March 2020 and February 2021 using validated reference-based sequencing workflow. We document the changing frequency of dominant variants of SARS-CoV-2 (from B.1 -> B.1.1.266 -> B.1.258 -> B.1.1.7) throughout the first year of the pandemic and list specific variants that could impact the diagnostic efficiency RT-qPCR assays. Moreover, our reference-based workflow provided evidence of superinfection in several samples, which may have contributed to one of the highest per capita numbers of COVID-19 cases and deaths during the first year of the pandemic in the Czech Republic.

Performance of Targeted Library Preparation Solutions for SARS-CoV-2 Whole Genome Analysis.

Single next-generation sequencing (NGS) proved to be an important tool for monitoring the SARS-CoV-2 outbreak at the global level Until today, thousands of SARS-CoV-2 genome sequences have been published at GISAID (Global Initiative on Sharing All Influenza Data) but only a portion are suitable for reliable variant analysis. Here we report on the comparison of three commercially available NGS library preparation kits. We discuss advantages and limitations from the perspective of required input sample quality and data quality for advanced SARS-CoV-2 genome analysis.

The development of Leishmania turanica in sand flies and competition with L. major.

BACKGROUND: In Central Asian foci of zoonotic cutaneous leishmaniases, mixed infections of Leishmania turanica and L. major have been found in a reservoir host (the great gerbil, Rhombomys opimus) as well as in the sand fly vector Phlebotomus papatasi, but hybrids between these two Leishmania species have never been reported. In addition, the role of sand fly species other than P. papatasi in L. turanica circulation is not clear. METHODS: In this work we compared the development of L. turanica in three sand fly species belonging to different subgenera. In addition, we studied experimental co-infections of sand flies by both Leishmania species using GFP transfected L. turanica (MRHO/MN/08/BZ18(GFP+)) and RFP transfected L. major (WHOM/IR/-/173-DsRED(RFP+)). The possibility of Leishmania genetic exchange during the vectorial part of the life cycle was studied using flow cytometry combined with immunofluorescent microscopy. RESULTS: Late-stage infections of L. turanica with frequent colonization of the stomodeal valve were observed in the specific vector P. (Phlebotomus) papatasi and in the permissive vector P. (Adlerius) arabicus. On the other hand, in P. sergenti (the specific vector of L. tropica), L. turanica promatigotes were present only until the defecation of bloodmeal remnants. In their natural vector P. papatasi, L. turanica and L. major developed similarly, and the spatiotemporal dynamics of localization in the sand fly gut was the same for both leishmania species. Fluorescence microscopy in combination with FACS analyses did not detect any L. major / L. turanica hybrids in the experimental co-infection of P. papatasi and P. duboscqi. CONCLUSION: Our data provide new insight into the development of different leishmania parasite species during a mixed infection in the sand fly gut. Despite the fact that both Leishmania species developed well in P. papatasi and P. duboscqi and did not outcompete each other, no genetic exchange was found. However, the ability of L. turanica to establish late-stage infections in these specific vectors of L. major suggests that the lipophosphoglycan of this species must be identical or similar to that of L. major.