NanoSPC: a scalable, portable, cloud compatible viral nanopore metagenomic data processing pipeline.

Metagenomic sequencing combined with Oxford Nanopore Technology has the potential to become a point-of-care test for infectious disease in public health and clinical settings, providing rapid diagnosis of infection, guiding individual patient management and treatment strategies, and informing infection prevention and control practices. However, publicly available, streamlined, and reproducible pipelines for analyzing Nanopore metagenomic sequencing data are still lacking. Here we introduce NanoSPC, a scalable, portable and cloud compatible pipeline for analyzing Nanopore sequencing data. NanoSPC can identify potentially pathogenic viruses and bacteria simultaneously to provide comprehensive characterization of individual samples. The pipeline can also detect single nucleotide variants and assemble high quality complete consensus genome sequences, permitting high-resolution inference of transmission. We implement NanoSPC using Nextflow manager within Docker images to allow reproducibility and portability of the analysis. Moreover, we deploy NanoSPC to our scalable pathogen pipeline platform, enabling elastic computing for high throughput Nanopore data on HPC cluster as well as multiple cloud platforms, such as Google Cloud, Amazon Elastic Computing Cloud, Microsoft Azure and OpenStack. Users could either access our web interface (https://nanospc.mmmoxford.uk) to run cloud-based analysis, monitor process, and visualize results, as well as download Docker images and run command line to analyse data locally.

Catwalk: identifying closely related sequences in large microbial sequence databases.

There is a need to identify microbial sequences that may form part of transmission chains, or that may represent importations across national boundaries, amidst large numbers of SARS-CoV-2 and other bacterial or viral sequences. Reference-based compression is a sequence analysis technique that allows both a compact storage of sequence data and comparisons between sequences. Published implementations of the approach are being challenged by the large sample collections now being generated. Our aim was to develop a fast software detecting highly similar sequences in large collections of microbial genomes, including millions of SARS-CoV-2 genomes. To do so, we developed Catwalk, a tool that bypasses bottlenecks in the generation, comparison and in-memory storage of microbial genomes generated by reference mapping. It is a compiled solution, coded in Nim to increase performance. It can be accessed via command line, rest api or web server interfaces. We tested Catwalk using both SARS-CoV-2 and Mycobacterium tuberculosis genomes generated by prospective public-health sequencing programmes. Pairwise sequence comparisons, using clinically relevant similarity cut-offs, took about 0.39 and 0.66 µs, respectively; in 1 s, between 1 and 2 million sequences can be searched. Catwalk operates about 1700 times faster than, and uses about 8 % of the RAM of, a Python reference-based compression and comparison tool in current use for outbreak detection. Catwalk can rapidly identify close relatives of a SARS-CoV-2 or M. tuberculosis genome amidst millions of samples.

Mycobacterium tuberculosis transmission in Birmingham, UK, 2009-19: An observational study.

Background: Over 10-years of whole-genome sequencing (WGS) of Mycobacterium tuberculosis in Birmingham presents an opportunity to explore epidemiological trends and risk factors for transmission in new detail. Methods: Between 1st January 2009 and 15th June 2019, we obtained the first WGS isolate from every patient resident in a postcode district covered by Birmingham's centralised tuberculosis service. Data on patients' sex, country of birth, social risk-factors, anatomical locus of disease, and strain lineage were collected. Poisson harmonic regression was used to assess seasonal variation in case load and a mixed-effects multivariable Cox proportionate hazards model was used to assess risk factors for a future case arising in clusters defined by a 5 single nucleotide polymorphism (SNP) threshold, and by 12 SNPs in a sensitivity analysis. Findings: 511/1653 (31%) patients were genomically clustered with another. A seasonal variation in diagnoses was observed, peaking in spring, but only among clustered cases. Risk-factors for a future clustered case included UK-birth (aHR=2·03 (95%CI 1·35-3·04), p < 0·001), infectious (pulmonary/laryngeal/miliary) tuberculosis (aHR=3·08 (95%CI 1·98-4·78), p < 0·001), and M. tuberculosis lineage 3 (aHR=1·91 (95%CI 1·03-3·56), p = 0·041) and 4 (aHR=2·27 (95%CI 1·21-4·26), p = 0·011), vs. lineage 1. Similar results pertained to 12 SNP clusters, for which social risk-factors were also significant (aHR 1·72 (95%CI 1·02-2·93), p = 0·044). There was marked heterogeneity in transmission patterns between postcode districts. Interpretation: There is seasonal variation in the diagnosis of genomically clustered, but not non-clustered, cases. Risk factors for clustering include UK-birth, infectious forms of tuberculosis, and infection with lineage 3 or 4. Funding: Wellcome Trust, MRC, UKHSA.

Differential occupational risks to healthcare workers from SARS-CoV-2 observed during a prospective observational study.

We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. 1128/10,034 (11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household contact were at greatest risk (adjusted odds ratio [aOR] 4.82 [95%CI 3.45-6.72]). Higher rates of Covid-19 were seen in staff working in Covid-19-facing areas (22.6% vs. 8.6% elsewhere) (aOR 2.47 [1.99-3.08]). Controlling for Covid-19-facing status, risks were heterogenous across the hospital, with higher rates in acute medicine (1.52 [1.07-2.16]) and sporadic outbreaks in areas with few or no Covid-19 patients. Covid-19 intensive care unit staff were relatively protected (0.44 [0.28-0.69]), likely by a bundle of PPE-related measures. Positive results were more likely in Black (1.66 [1.25-2.21]) and Asian (1.51 [1.28-1.77]) staff, independent of role or working location, and in porters and cleaners (2.06 [1.34-3.15]).