Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing.

Mycobacterium tuberculosis whole-genome sequencing (WGS) is a powerful tool as it can provide data on population diversity, drug resistance, disease transmission, and mixed infections. Successful WGS is still reliant on high concentrations of DNA obtained through M. tuberculosis culture. Microfluidics technology plays a valuable role in single-cell research but has not yet been assessed as a bacterial enrichment strategy for culture-free WGS of M. tuberculosis. In a proof-of-principle study, we evaluated the use of Capture-XT, a microfluidic lab-on-chip cleanup and pathogen concentration platform to enrich M. tuberculosis bacilli from clinical sputum specimens for downstream DNA extraction and WGS. Three of the four (75%) samples processed by the microfluidics application passed the library preparation quality control, compared to only one of the four (25%) samples not enriched by the microfluidics M. tuberculosis capture application. WGS data were of sufficient quality, with mapping depth of ≥25× and 9 to 27% of reads mapping to the reference genome. These results suggest that microfluidics-based M. tuberculosis cell capture might be a promising method for M. tuberculosis enrichment in clinical sputum samples, which could facilitate culture-free M. tuberculosis WGS. IMPORTANCE Diagnosis of tuberculosis is effective using molecular methods; however, a comprehensive characterization of the resistance profile of Mycobacterium tuberculosis often requires culturing and phenotypic drug susceptibility testing or culturing followed by whole-genome sequencing (WGS). The phenotypic route can take anywhere from 1 to >3 months to result, by which point the patient may have acquired additional drug resistance. The WGS route is a very attractive option; however, culturing is the rate-limiting step. In this original article, we provide proof-of-principle evidence that microfluidics-based cell capture can be used on high-bacillary-load clinical samples for culture-free WGS.

A Novel Microfluidic Dielectrophoresis Technology to Enable Rapid Diagnosis of Mycobacteria tuberculosis in Clinical Samples.

To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated with bacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing.

Synthesis of Polyanionic C5-Modified 2'-Deoxyuridine and 2'-Deoxycytidine-5'-Triphosphates and Their Properties as Substrates for DNA Polymerases.

Modified 2'-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.

Restriction of Retrotransposon Mobilization in Schizosaccharomyces pombe by Transcriptional Silencing and Higher-Order Chromatin Organization.

Uncontrolled propagation of retrotransposons is potentially detrimental to host genome integrity. Therefore, cells have evolved surveillance mechanisms to restrict the mobility of these elements. In Schizosaccharomyces pombe the Tf2 LTR retrotransposons are transcriptionally silenced and are also clustered in the nucleus into structures termed Tf bodies. Here we describe the impact of silencing and clustering on the mobility of an endogenous Tf2 element. Deletion of genes such as set1(+) (histone H3 lysine 4 methyltransferase) or abp1(+) (CENP-B homolog) that both alleviate silencing and clustering, result in a corresponding increase in mobilization. Furthermore, expression of constitutively active Sre1, a transcriptional activator of Tf2 elements, also alleviates clustering and induces mobilization. In contrast, clustering is not disrupted by loss of the HIRA histone chaperone, despite high levels of expression, and in this background, mobilization frequency is only marginally increased. Thus, mutations that compromise transcriptional silencing but not Tf bodies are insufficient to drive mobilization. Furthermore, analyses of mutant alleles that separate the transcriptional repression and clustering functions of Set1 are consistent with control of Tf2 propagation via a combination of silencing and spatial organization. Our results indicate that host surveillance mechanisms operate at multiple levels to restrict Tf2 retrotransposon mobilization.

Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organisation.

Maintenance of the correct level and organisation of nucleosomes is crucial for genome function. Here, we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in the maintenance of nucleosome architecture in fission yeast. Cells lacking abo1(+) experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide.

The fission yeast HIRA histone chaperone is required for promoter silencing and the suppression of cryptic antisense transcripts.

The assembly of nucleosomes by histone chaperones is an important component of transcriptional regulation. Here, we have assessed the global roles of the HIRA histone chaperone in Schizosaccharomyces pombe. Microarray analysis indicates that inactivation of the HIRA complex results in increased expression of at least 4% of fission yeast genes. HIRA-regulated genes overlap with those which are normally repressed in vegetatively growing cells, such as targets of the Clr6 histone deacetylase and silenced genes located in subtelomeric regions. HIRA is also required for silencing of all 13 intact copies of the Tf2 long terminal repeat (LTR) retrotransposon. However, the role of HIRA is not restricted to bona fide promoters, because HIRA also suppresses noncoding transcripts from solo LTR elements and spurious antisense transcripts from cryptic promoters associated with transcribed regions. Furthermore, the HIRA complex is essential in the absence of the quality control provided by nuclear exosome-mediated degradation of illegitimate transcripts. This suggests that HIRA restricts genomic accessibility, and consistent with this, the chromosomes of cells lacking HIRA are more susceptible to genotoxic agents that cause double-strand breaks. Thus, the HIRA histone chaperone is required to maintain the protective functions of chromatin.