Early detection and surveillance of SARS-CoV-2 genomic variants in wastewater using COJAC.

The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK.

DeepSARS: simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2.

BACKGROUND: The continued spread of SARS-CoV-2 and emergence of new variants with higher transmission rates and/or partial resistance to vaccines has further highlighted the need for large-scale testing and genomic surveillance. However, current diagnostic testing (e.g., PCR) and genomic surveillance methods (e.g., whole genome sequencing) are performed separately, thus limiting the detection and tracing of SARS-CoV-2 and emerging variants. RESULTS: Here, we developed DeepSARS, a high-throughput platform for simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2 by the integration of molecular barcoding, targeted deep sequencing, and computational phylogenetics. DeepSARS enables highly sensitive viral detection, while also capturing genomic diversity and viral evolution. We show that DeepSARS can be rapidly adapted for identification of emerging variants, such as alpha, beta, gamma, and delta strains, and profile mutational changes at the population level. CONCLUSIONS: DeepSARS sets the foundation for quantitative diagnostics that capture viral evolution and diversity. DeepSARS uses molecular barcodes (BCs) and multiplexed targeted deep sequencing (NGS) to enable simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2. Image was created using Biorender.com .

Quantification of the spread of SARS-CoV-2 variant B.1.1.7 in Switzerland.

BACKGROUND: In December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40-80 % (Volz et al., 2021; Leung et al., 2021; Davies et al., 2021). AIM: This study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland. METHODS: We generated whole genome sequences from 11.8 % of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant's transmission fitness advantage on a national and a regional scale. RESULTS: We estimate B.1.1.7 had a transmission fitness advantage of 43-52 % compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07-1.41] from 01 January until 17 January 2021 and 1.18 [1.06-1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00-1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021. CONCLUSION: The observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2-3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.

Innovative Tumor Tissue Dissection Tool for Molecular Oncology Diagnostics.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-µm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation.

Plasma-based longitudinal mutation monitoring as a potential predictor of disease progression in subjects with adenocarcinoma in advanced non-small cell lung cancer.

BACKGROUND: Identifying and tracking somatic mutations in cell-free DNA (cfDNA) by next-generation sequencing (NGS) has the potential to transform the clinical management of subjects with advanced non-small cell lung cancer (NSCLC). METHODS: Baseline tumor tissue (n = 47) and longitudinal plasma (n = 445) were collected from 71 NSCLC subjects treated with chemotherapy. cfDNA was enriched using a targeted-capture NGS kit containing 197 genes. Clinical responses to treatment were determined using RECIST v1.1 and correlations between changes in plasma somatic variant allele frequencies and disease progression were assessed. RESULTS: Somatic variants were detected in 89.4% (42/47) of tissue and 91.5% (407/445) of plasma samples. The most commonly mutated genes in tissue were TP53 (42.6%), KRAS (25.5%), and KEAP1 (19.1%). In some subjects, the allele frequencies of mutations detected in plasma increased 3-5 months prior to disease progression. In other cases, the allele frequencies of detected mutations declined or decreased to undetectable levels, indicating clinical response. Subjects with circulating tumor DNA (ctDNA) levels above background had significantly shorter progression-free survival (median: 5.6 vs 8.9 months, respectively; log-rank p = 0.0183). CONCLUSION: Longitudinal monitoring of mutational changes in plasma has the potential to predict disease progression early. The presence of ctDNA mutations during first-line treatment is a risk factor for earlier disease progression in advanced NSCLC.

A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins.

UNLABELLED: Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5' rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad. IMPORTANCE: Next-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAV in vivo, combined in cellulae and in silico studies will help to forward the understanding of the unique, bipartite AAV life cycle.

Exome sequencing helped the fine diagnosis of two siblings afflicted with atypical Timothy syndrome (TS2).

BACKGROUND: Long-QT syndrome (LQTS) causes a prolongation of the QT-interval in the ECG leading to life threatening tachyarrhythmia and ventricular fibrillation. One atypical form of LQTS, Timothy syndrome (TS), is associated with syndactyly, immune deficiency, cognitive and neurological abnormalities as well as distinct cranio-facial abnormalities. CASE PRESENTATION: On a family with both children diagnosed with clinical LQTS, we performed whole exome sequencing to comprehensively screen for causative mutations after a targeted candidate gene panel screen for Long-QT syndrome target genes failed to identify any underlying genetic defect. Using exome sequencing, we identified in both affected children, a p.402G > S mutation in exon 8 of the CACNA1C gene, a voltage-dependent Ca2+ channel. The mutation was inherited from their father, a mosaic mutation carrier. Based on this molecular finding and further more careful clinical examination, we refined the diagnosis to be Timothy syndrome (TS2) and thereby were able to present new therapeutic approaches. CONCLUSIONS: Our study highlights the difficulties in accurate diagnosis of patients with rare diseases, especially those with atypical clinical manifestation. Such challenge could be addressed with the help of comprehensive and unbiased mutation screening, such as exome sequencing.

Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity.

Plague is a pandemic human invasive disease caused by the bacterial agent Yersinia pestis. We here report a comparison of 17 whole genomes of Y. pestis isolates from global sources. We also screened a global collection of 286 Y. pestis isolates for 933 SNPs using Sequenom MassArray SNP typing. We conducted phylogenetic analyses on this sequence variation dataset, assigned isolates to populations based on maximum parsimony and, from these results, made inferences regarding historical transmission routes. Our phylogenetic analysis suggests that Y. pestis evolved in or near China and spread through multiple radiations to Europe, South America, Africa and Southeast Asia, leading to country-specific lineages that can be traced by lineage-specific SNPs. All 626 current isolates from the United States reflect one radiation, and 82 isolates from Madagascar represent a second radiation. Subsequent local microevolution of Y. pestis is marked by sequential, geographically specific SNPs.

Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus.

A small number of clonal lineages dominates the global population structure of methicillin-resistant Staphylococcus aureus (MRSA), resulting in the concept that MRSA has emerged on a few occasions after penicillinase-stable beta-lactam antibiotics were introduced to clinical practice, followed by intercontinental spread of individual clones. We investigated the evolutionary history of an MRSA clone (ST5) by mutation discovery at 108 loci (46 kb) within a global collection of 135 isolates. The SNPs that were ascertained define a radial phylogenetic structure within ST5 consisting of at least 5 chains of mutational steps that define geographically associated clades. These clades are not concordant with previously described groupings based on staphylococcal protein A gene (spa) typing. By mapping the number of independent imports of the staphylococcal cassette chromosome methicillin-resistance island, we also show that import has occurred on at least 23 occasions within this single sequence type and that the progeny of such recombinant strains usually are distributed locally rather than globally. These results provide strong evidence that geographical spread of MRSA over long distances and across cultural borders is a rare event compared with the frequency with which the staphylococcal cassette chromosome island has been imported.