Bacterial DNA enrichment for low-inoculum fracture-related infection diagnostic using high-throughput sequencing.

One of the main barriers for the implementation of metagenomic sequencing in routine diagnosis of infectious diseases is the presence of host DNA. While several enrichment methods are likely to overcome this issue, their effectiveness for specimens such as bone in the case of chronic infections remains to be determined. We compared the relevance of two methods for bacterial DNA enrichment when compared to a reference protocol during pretreatment of bone samples from fracture-related infections before HTS by both Illumina Miseq and Oxford Nanopore Technology (ONT). The bacterial/human DNA ratio was higher for either protocols than the reference technique (p = 0.00012), without any significant difference between them. HTS sensitivity over culture ranged from 21.7 % to 85 %. The ability of the studied protocols to improve the bacterial/human DNA ratio depends on the sequencing technique employed. In this context, there is room for improvement in enhancing the sensitivity of HTS for diagnostic purpose.

Methanobrevibacter massiliense and Pyramidobacter piscolens Co-Culture Illustrates Transkingdom Symbiosis.

Among oral microbiota methanogens, Methanobrevibacter massiliense (M. massiliense) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii. M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens (P. piscolens) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens, as non-motile, 1-2-µm-long and 0.6-0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies.

High Throughput SARS-CoV-2 Genome Sequencing from 384 Respiratory Samples Using the Illumina COVIDSeq Protocol.

The emergence of the Coronavirus Disease 2019 (COVID-19) pandemic has fostered the use of high-throughput techniques to sequence the entire severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and track its evolution. The present study proposes a rapid and relatively less expensive sequencing protocol for 384 samples by adapting the use of an Illumina NovaSeq library to an Illumina MiSeq flow cell instrument. The SARS-CoV-2 genome sequences obtained with Illumina NovaSeq and those obtained using MiSeq instruments were compared with the objective to validate the new, modified protocol. A total of 356 (94.6%) samples yielded interpretable sequences using the modified Illumina COVIDSeq protocol, with an average coverage of 91.6%. By comparison, 357 (94.9%) samples yielded interpretable sequences with the standard COVIDSeq protocol, with an average coverage of 95.6%. Our modified COVIDSeq protocol could save 14,155 euros per run and yield results from 384 samples in 53.5 h, compared to four times 55.5 h with the standard Illumina MiSeq protocol. The modified COVIDSeq protocol thus provides high quality results comparable to those obtained with the standard COVIDSeq protocol, four times faster, while saving money.

Genomic Epidemiology of SARS-CoV-2 in Urban Settings in Senegal.

We used whole genome sequencing to identify and analyze mutations in SARS-CoV-2 in urban settings during the deadliest wave of the COVID-19 epidemic-from March to April 2021-in Senegal. Nasopharyngeal samples testing positive for SARS-CoV-2 were sequenced on the Illumina NovaSeq 6000 sequencing system using the COVIDSeq protocol. A total of 291 genotypable consensus genome sequences were obtained. Phylogenetic analyses grouped the genomes into 16 distinct PANGOLIN lineages. The major lineage was B.1.1.420, despite circulation of the Alpha variant of concern (VOC). A total of 1125 different SNPs, identified relative to the Wuhan reference genome, were detected. These included 13 SNPs in non-coding regions. An average density of 37.2 SNPs per 1000 nucleotides was found, with the highest density occurring in ORF10. This analysis allowed, for the first time, the detection of a Senegalese SARS-CoV-2 strain belonging to the P.1.14 (GR/20J, Gamma V3) sublineage of the Brazilian P.1 lineage (or Gamma VOC). Overall, our results highlight substantial SARS-CoV-2 diversification in Senegal during the study period.

Simultaneous SARS-CoV-2 Genome Sequencing of 384 Samples on an Illumina MiSeq Instrument through Protocol Optimization.

In the present study, we propose a high-throughput sequencing protocol using aNextera XT Library DNA kit on an Illumina MiSeq instrument. We made major modifications to this library preparation in order to multiplex 384 samples in a single Illumina flow cell. To validate our protocol, we compared the sequences obtained with the modified Illumina protocol to those obtained with the GridION Nanopore protocol. For the modified Illumina protocol, our results showed that 94.9% (357/376) of the sequences were interpretable, with a viral genome coverage between 50.5% and 99.9% and an average depth of 421×. For the GridION Nanopore protocol, 94.6% (356/376) of the sequences were interpretable, with a viral genome coverage between 7.0% and 98.6% and an average depth of 2123×. The modified Illumina protocol allows for gaining EUR 4744 and returning results of 384 samples in 53.5 h versus four times 55.5 h with the standard Illumina protocol. Our modified MiSeq protocol yields similar genome sequence data as the GridION Nanopore protocol and has the advantage of being able to handle four times more samples simultaneously and hence is much less expensive.