{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"medRxiv"}],"indexed":{"date-parts":[[2025,12,20]],"date-time":"2025-12-20T18:27:23Z","timestamp":1766255243205,"version":"3.48.0"},"posted":{"date-parts":[[2025,12,18]]},"group-title":"Infectious Diseases (except HIV\/AIDS)","reference-count":28,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T00:00:00Z","timestamp":1766016000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background:<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    (\n                    <jats:italic>Mtb<\/jats:italic>\n                    ) genomic epidemiology often relies on culturing patient sputum, a time and labor-intensive process. Hybrid capture approaches have been successfully used to enrich\n                    <jats:italic>Mtb<\/jats:italic>\n                    DNA from complex clinical samples, yet the accuracy of variant identification from captured samples has not been systematically evaluated.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods:<\/jats:title>\n                  <jats:p>\n                    We created artificial strain mixtures of two well-characterized\n                    <jats:italic>Mtb<\/jats:italic>\n                    isolates such that the minor strain comprised 0-50% of\n                    <jats:italic>Mtb<\/jats:italic>\n                    DNA and serially diluted the\n                    <jats:italic>Mtb<\/jats:italic>\n                    DNA into human DNA to simulate diagnostic samples with different sputum smear burdens (32 samples). We also prospectively collected paired\n                    <jats:italic>Mtb<\/jats:italic>\n                    diagnostic cultures and sputum submitted to a national diagnostic laboratory (7 sample pairs). We performed hybrid capture and Illumina whole genome sequencing for all samples. For the artificial strain mixtures, we measured hybrid capture efficiency, the percentage of total reads mapping to\n                    <jats:italic>Mtb<\/jats:italic>\n                    , and performance of fixed and minority variant identification. For the diagnostic samples, we compared the number, identity of, and minor allele frequencies of minority variants identified in the cultured and hybrid captured samples.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results:<\/jats:title>\n                  <jats:p>\n                    In the artificial strain mixture experiment, hybrid capture efficiency was 97% when\n                    <jats:italic>Mtb<\/jats:italic>\n                    comprised 0.01% of the input DNA. Single nucleotide polymorphism (SNP) identification via hybrid capture had a sensitivity \u2265 91% and precision \u2265 97% for\n                    <jats:italic>Mtb<\/jats:italic>\n                    lineages 4.1.2.1 and 4.9, excluding PE\/PPE genes, when\n                    <jats:italic>Mtb<\/jats:italic>\n                    comprised 0.01% of input DNA. Observed minor allele frequencies were closely correlated (r=0.60 to r=0.79, p &lt; 0.001) with input minor allele frequences across all dilutions. Among paired diagnostic samples, hybrid capture efficiency was high, 95%. However, four of the seven captured sputa samples were overwhelmed with\n                    <jats:italic>Pseudomonas<\/jats:italic>\n                    contamination, which comprised &gt;25% of sequence reads. We did not detect a significant difference in the number of minority variants identified in cultured (median: 14, range: 9-23) and hybrid captured samples (median: 22 variants, range 4-328, p=0.2) and minor allele frequences were correlated (r = 0.95, p &lt; 0.001)\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions:<\/jats:title>\n                  <jats:p>\n                    Hybrid capture of diagnostic sputa samples efficiently generates accurate\n                    <jats:italic>Mtb<\/jats:italic>\n                    whole genome sequences and minority variant calls. Hybrid capture may offer an alternative to culture-based sequencing that could extend the coverage of genomic epidemiology studies.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Impact Statement:<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    (\n                    <jats:italic>Mtb<\/jats:italic>\n                    ) genomic epidemiology often relies on culturing patient sputa, which is time and labor-intensive. Hybrid capture approaches enrich\n                    <jats:italic>Mtb<\/jats:italic>\n                    DNA from complex clinical samples, yet the efficiency and accuracy of identification of both consensus and minority variants from captured samples has not been systematically evaluated. To address these questions, we assessed the accuracy of hybrid capture both in experimental strain mixtures of well-characterized\n                    <jats:italic>Mtb<\/jats:italic>\n                    isolates and in clinical diagnostic samples. We found that hybrid capture of sputa samples can be used to efficiently sequence\n                    <jats:italic>Mtb<\/jats:italic>\n                    DNA from complex mixtures and that variant identification is highly accurate. Our results suggest that hybrid capture may offer an alternative to culture-based sequencing that could extend the coverage of genomic epidemiology studies.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2025.12.16.25342406","type":"posted-content","created":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T20:45:16Z","timestamp":1766090716000},"source":"Crossref","is-referenced-by-count":0,"title":["Optimizing culture-free approaches to recover high quality\n                  <i>M. tuberculosis<\/i>\n                  genomic variation"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0065-2204","authenticated-orcid":false,"given":"Katharine S.","family":"Walter","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paulo C\u00e9sar Pereira","family":"Dos Santos","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Allison","family":"Carey","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Salika M.","family":"Shakir","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Caroline","family":"Colijn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ted","family":"Cohen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Barun","family":"Mathema","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6665-6825","authenticated-orcid":false,"given":"Julio","family":"Croda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jason R.","family":"Andrews","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025122010250712000_2025.12.16.25342406v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-019-0214-5"},{"key":"2025122010250712000_2025.12.16.25342406v1.2","unstructured":"Mukamolova G V , Turapov O , Malkin J , Woltmann G , Barer MR . 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