Decontamination

Patients meeting the clinical eligibility criteria were asked to provide three sputum specimens over a 2-day period (two spot samples and one obtained in the morning) (Fig. 1). In a random fashion, two of the three samples were processed with N-acetyl-l-cysteine and sodium hydroxide (NALC–NaOH),14 followed by centrifugation, and then were resuspended in 1.5 ml of phosphate buffer and subjected to microscopy with Ziehl–Neelsen staining, and cultivation on solid medium (egg-based Löwenstein–Jensen15 or 7H11,16 (link) with the latter medium used only in Durban) and liquid medium (BACTECMGIT [mycobacteria growth indicator tube] 960 culture; BD Microbiology Systems), and the MTB/RIF test. The third sputum sample was tested directly by Ziehl–Neelsen microscopy and the MTB/RIF test without NALC–NaOH decontamination.
The first positive culture from each specimen underwent confirmation of M. tuberculosis species by MPT64 antigen detection (Capilia TB, Tauns Laboratories)17 (link) and indirect drug-susceptibility testing with the proportion method on Löwenstein–Jensen medium (for sites in Lima, Durban, and Baku) or MGIT SIRE18 (for sites in Cape Town and Mumbai). For three sites, conventional nucleic acid–amplification testing was carried out on DNA that was extracted from the NALC–NaOH centrifugation pellet of the first sputum sample with the use of Cobas Amplicor MTB (Roche) (in Cape Town and Mumbai) or ProbeTec ET MTB Complex Direct Detection Assay (BD) (in Baku), according to the manufacturer's instructions. At three sites, drug-resistant genotyping was carried out by line-probe assay with the use of the Geno-type MTBDRplus assay (Hain Lifescience) performed from culture isolates (in Baku) or from the NALC–NaOH pellet of the second sputum sample (in Cape Town and Durban), according to the manufacturer's instructions, except that smear-negative specimens were also tested.
All participating laboratories were quality-assured reference laboratories. Study laboratories for four sites were located within 5 km of the enrollment clinic and tested samples within 2 days after collection. Sputum samples from Baku were shipped to the German National Reference Laboratory in Borstel for testing 1 to 5 days after collection.
Repeat tuberculosis analyses (smear, culture, MTB/RIF test, radiography, and clinical workup) were performed in patients who had smear- and culture-negative samples if the MTB/RIF test or other nucleic acid–amplification test was positive or if the patient was selected by the central database as a random control for follow-up. The final diagnosis for patients undergoing repeat analyses was established on the basis of conventional laboratory results and clinical information by clinical review committees composed of three local tuberculosis clinicians. HIV results were obtained by review of clinical records and were available for only a subgroup of patients. Bias was minimized through blinding, since technicians performing molecular and reference tests were not aware of the results of other tests. The interpretation of data from MTB/RIF tests was software-based and independent of the user. Clinical teams and review committees did not have access to nucleic acid–amplification test results. All study coordinators received lists of patients for follow-up but not the reasons for follow-up.

New RNA-Seq data was generated for 23 gnathostome species using Illumina MiSeq (2x250 bp) and HiSeq2000 (2x50 bp, 2x100 bp) technologies. Available RNA-Seq data were downloaded from NCBI SRA. Transcriptomes were assembled de novo with Trinity or MIRA. Species names and accession numbers are available in Supplementary Table S10.
Nuclear datasets were assembled using a new pipeline summarized in Supplementary Fig. 2.
Briefly, proteomes of 21 vertebrate genomes (ENSEMBL) were grouped into ortholog
clusters and those not containing data for all major jawed vertebrate lineages
were discarded. The resulting 11,656 protein clusters were aligned and positions
of unreliable homology removed. To identify and resolve paralogy issues, we
implemented a paralog-splitting pipeline based on gene trees. The obtained 9,852
ortholog clusters were complemented with new genomes and transcriptomes using
the software Forty-Two (https://bitbucket.org/dbaurain/42/). Several decontamination
steps were carried out. Any sequence contamination from non-vertebrates and
human was detected by BLAST and eliminated. We searched for cross-contamination
that can arise during library preparation using gene trees, and removed
contaminants based on expression data. After eliminating overlapping redundant
sequences that were too divergent, we filtered out incomplete or short sequences
and alignments, leading to 7,687 genes. The paralogy splitting procedure was
repeated to resolve any paralogy caused by the addition of new species, and gene
alignments were classified into three datasets that contained zero (NoDP), one
(1DP) and two or more (2DP) deep paralogs. Sequence stretches with unusually low
similarity (usually due to frame shifts) were masked with HMM-cleaner (R.
Poujol) and alignments were trimmed. For each gene, we used SCaFoS30 to merge conspecific sequences and
resolve putative remaining paralogy. A third decontamination step used extremely
long branches estimated on a fixed reference tree as proxy for
contamination.
Mitochondrial datasets were assembled from mitogenomes available at NCBI with a taxon sampling mirroring the nuclear datasets plus a few additional species to reduce long-branch attraction artefacts expected in mitogenomic trees (Supplementary Table 11). The resulting alignments consisted of 106 species (2,773 amino acid positions) and 95 species (2,866 amino acid positions) after removing the fastest evolving species.

Male golden Syrian hamsters at 4–5 weeks old were obtained from Laboratory Animal Services Centre, Chinese University of Hong Kong. The hamsters were originally imported from Harlan (Envigo), UK in 1998. All experiments were performed at the BSL-3 core facility, LKS Faculty of Medicine, The University of Hong Kong. The animals were randomized from different litters into experimental groups, and the animals were acclimatized at the BSL3 facility for 4–6 days prior to the experiments. The study protocol have been reviewed and approved by the Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong (CULATR # 5323–20). Experiments were performed in compliance with all relevant ethical regulations. For challenge studies, hamsters were anesthetized by ketamine(150mg/kg) and xylazine (10mg/kg) via intra-peritoneal injection and were intra-nasally inoculated with 8 × 10⁴ TCID₅₀ of SARS-CoV-2 in 80 μL DMEM. On days 2, 5, 7, three hamsters were euthanized by intra-peritoneal injection of pentobarbital at 200mg/kg. No blinding was done and a sample size of three animals was selected to assess the level of variation between animals. Lungs (left) and one kidney were collected for viral load determination and were homogenized in 1mL PBS. Brain, nasal turbinate, lungs (right, liver, heart, spleen, duodenum, and kidney were fixed in 4% paraformaldehyde for histopathological examination. To collect fecal samples, hamsters were transferred to a new cage one day in advance and fresh fecal samples (10 pieces) were collected for quantitative real-time RT-PCR and TCID50 assay. To evaluate SARS-CoV-2 transmissibility by direct contact, donor hamsters were anesthetized and inoculated with 8 × 10⁴ TCID₅₀ of SARS-CoV-2. On 1 dpi or on 6 dpi, one inoculated donor was transferred to co-house with one naïve hamster in a clean cage and co-housing of the animals continued for at least 13 days. Experiments were repeated with three pairs of donors: direct contact at 1:1 ratio^{31 (link),32 (link)}. Body weight and clinical signs of the animals were monitored daily. To evaluate SARS-CoV-2 transmissibility via aerosols, one naïve hamster was exposed to one inoculated donor hamster in two adjacent stainless steel wired cages on 1 dpi for 8 hours (Extended Data Fig. 3). DietGel®76A (ClearH₂O®) was provided to the hamsters during the 8-hour exposure. Exposure was done by holding the animals inside individually ventilated cages (IsoCage N, Techniplast) with 70 air changes per hour. Experiments were repeated with three pairs of donors: aerosol contact at 1:1 ratio. After exposure, the animals were single-housed in separate cages and were continued monitored for 14 days. To evaluate transmission potential of SARS-CoV-2 virus via fomites, three naïve fomite contact hamsters were each introduced to a soiled donor cage on 2 dpi. The fomite contact hamsters were single-housed for 48 hours inside the soiled cages and then were each transferred to a new cage on 4 dpi of the donor. All animals were continued monitored for 14 days. For nasal wash collection, hamsters were anesthetized by ketamine (100mg/kg) and xylazine (10mg/kg) via intra-peritoneal injection and 160 μL of PBS containing 0.3% BSA was used to collect nasal washes from both nostrils of each animal. Collected nasal washes were diluted 1:1 by volume and aliquoted for TCID₅₀ assay in Vero E6 cells and for quantitative real-time RT-PCR. The contact hamster were handled first followed by surface decontamination using 1% virkon and handling of the donor hamster.