A critical spotlight on the paradigms of FFPE-DNA sequencing.

In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called 'ERROR-FFPE-DNA' that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.

Molecular profiling of advanced soft-tissue sarcomas: the MULTISARC randomized trial.

BACKGROUND: Soft-tissue sarcomas (STS) represent a heterogeneous group of rare tumors including more than 70 different histological subtypes. High throughput molecular analysis (next generation sequencing exome [NGS]) is a unique opportunity to identify driver mutations that can change the usual one-size-fits-all treatment paradigm to a patient-driven therapeutic strategy. The primary objective of the MULTISARC trial is to assess whether NGS can be conducted for a large proportion of metastatic STS participants within a reasonable time, and, secondarily to determine whether a NGS-guided therapeutic strategy improves participant's outcome. METHODS: This is a randomized, multicentre, phase II/III trial inspired by the design of umbrella and biomarker-driven trials. The setting plans up to 17 investigational centres across France and the recruitment of 960 participants. Participants aged at least 18 years, with unresectable locally advanced and/or metastatic STS confirmed by the French sarcoma pathological reference network, are randomized according to 1:1 allocation ratio between the experimental arm "NGS" and the standard "No NGS". NGS will be considered feasible if (i) NGS results are available and interpretable, and (ii) a report of exome sequencing including a clinical recommendation from a multidisciplinary tumor board is provided to investigators within 7 weeks from reception of the samples on the biopathological platform. A feasibility rate of more than 70% is expected (null hypothesis: 70% versus alternative hypothesis: 80%). In terms of care, participants randomized in "No NGS" arm and who fail treatment will be able to switch to the NGS arm at the request of the investigator. DISCUSSION: The MULTISARC trial is a prospective study designed to provide high-level evidence to support the implementation of NGS in routine clinical practice for advanced STS participants, on a large scale. TRIAL REGISTRATION: clinicaltrial.gov NCT03784014 .

Heterogeneity of SARS-CoV-2 virus produced in cell culture revealed by shotgun proteomics and supported by genome sequencing.

COVID-19 is the most disturbing pandemic of the past hundred years. Its causative agent, the SARS-CoV-2 virus, has been the subject of an unprecedented investigation to characterize its molecular structure and intimate functioning. While markers for its detection have been proposed and several diagnostic methodologies developed, its propensity to evolve and evade diagnostic tools and the immune response is of great concern. The recent spread of new variants with increased infectivity requires even more attention. Here, we document how shotgun proteomics can be useful for rapidly monitoring the evolution of the SARS-CoV-2 virus. We evaluated the heterogeneity of purified SARS-CoV-2 virus obtained after culturing in the Vero E6 cell line. We found that cell culture induces significant changes that are translated at the protein level, such changes being detectable by tandem mass spectrometry. Production of viral particles requires careful quality control which can be easily performed by shotgun proteomics. Although considered relatively stable so far, the SARS-CoV-2 genome turns out to be prone to frequent variations. Therefore, the sequencing of SARS-CoV-2 variants from patients reporting only the consensus genome after its amplification would deserve more attention and could benefit from more in-depth analysis of low level but crystal-clear signals, as well as complementary and rapid analysis by shotgun proteomics.

Definition of a concentration and RNA extraction protocol for optimal whole genome sequencing of SARS-CoV-2 in wastewater (ANRS0160).

Monitoring the presence of RNA from emerging pathogenic viruses, such as SARS-CoV-2, in wastewater (WW) samples requires suitable methods to ensure an effective response. Genome sequencing of WW is one of the crucial methods, but it requires high-quality RNA in sufficient quantities, especially for monitoring emerging variants. Consequently, methods for viral concentration and RNA extraction from WW samples have to be optimized before sequencing. The purpose of this study was to achieve high coverage (≥ 90 %) and sequencing depth (at least ≥200×) even for low initial RNA concentrations (< 105 genome copies (GC)/L) in WW. A further objective was to determine the range of SARS-CoV-2 RNA concentrations that allow high-quality sequencing, and the optimal sample volume for analysis. Ultrafiltration (UF) methods were used to concentrate viral particles from large influent samples (up to 500 mL). An RNA extraction protocol using silica beads, neutral phenol-chloroform treatment, and a PCR inhibitor removal kit was chosen for its effectiveness in extracting RNA and eliminating PCR inhibitors, as well as its adaptability for use with large influent samples. Recovery rates ranged from 24 % to 63 % (N = 17) for SARS-CoV-2 naturally present in WW samples. 200 mL WW samples can be enough for UF concentration, as they showed high quality sequencing analyses with between 5 × 104 GC/L and 6 × 103 GC/L. Below 6 × 103 GC/L, high-quality sequencing was also achieved for ∼40 % of the samples using 500 mL of WW. Sequencing analysis for variant detection was performed on 200 mL WW samples with coverage of >95 % and sequencing depth of >1000×. Analyses revealed the predominance of variant EG.5, known as Eris (66 %-100 %). The use of UF methods in combination with a suitable RNA extraction protocol appear promising for sequencing enveloped viruses in WW in a context of viral emergence.

A comparison of high-throughput SARS-CoV-2 sequencing methods from nasopharyngeal samples.

The COVID-19 pandemic caused by the new Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to threaten public health and burden healthcare systems worldwide. Whole SARS-CoV-2 genome sequencing has become essential for epidemiological monitoring and identification of new variants, which could represent a risk of increased transmissibility, virulence, or resistance to vaccines or treatment. Different next-generation sequencing approaches are used in SARS-CoV-2 sequencing, although with different ability to provide whole genome coverage without gaps and to reliably detect new variants. In this study, we compared the performance of three target enrichment methods (two multiplex amplification methods and one hybridization capture) using nasopharyngeal swabs from infected individuals. We applied these target enrichment methods to the same set of nasopharyngeal samples (N = 93) in high-throughput mode. SARS-CoV-2 genome was obtained using short-read next-generation sequencing. We observed that each method has some advantages, such as high mapping rate (CleanPlex and COVIDSeq) or absence of systematic variant calling error (SureSelect) as well as their limitations such as suboptimal uniformity of coverage (CleanPlex), high cost (SureSelect) or supply shortages (COVIDSeq). Nevertheless, each of the three target enrichment kits tested in this study yielded acceptable results of whole SARS-CoV-2 genome sequencing and either of them can therefore be used in prospective programs of genomic surveillance of SARS-CoV-2. Genomic surveillance will be crucial to overcoming the ongoing pandemic of COVID-19, despite its successive waves and continually emerging variants.