Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes

The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing™) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200 000 to 300 000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments

Unique Features of a Highly Pathogenic Campylobacter jejuni Strain

Campylobacter jejuni, a major human enteric pathogen, exhibits significant strain-to-strain differences which result in differences in pathogenic potential. C. jejuni 81-176 is a highly virulent strain that exhibits unique pathogenic features and is used by many research laboratories. We have determined the nucleotide sequence of its genome and compared it to the genomes of other sequenced C. jejuni strains. We identified a number of unique genetic features which may confer specific metabolic and pathogenic properties on this strain. We have also identified regions of the C. jejuni genome that are hot spots for the integration of horizontally acquired genetic material. This information should help the understanding of the pathogenesis of C. jejuni and, in particular, the unique features of this highly pathogenic strain

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented. We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before). Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape

No barrier to care, yet disparities in the HIV care continuum in France: a nationwide population study

International audienceAbstract Objectives Even in an ‘optimal’ health system, patients’ characteristics may have an impact on their care. We investigated whether age, gender and place of birth have an impact in the HIV care continuum in France, a country with a universal free healthcare system. Methods We estimated differences in the 5 year restricted mean percentage of person-time spent (i) in care, (ii) receiving ART and (iii) on ART and virally suppressed among 2432 (30.2%) women, 3925 MSM (48.7%) and 1709 men who have sex with women (MSW; 21.2%) entering care in the Dat’AIDS French prospective cohort between 1 January 2013 and 31 December 2017. Trial registration: Clinicaltrials.gov reference NCT02898987. Results Men and women spent 85.6% and 82.8% of person-time on ART and 69.9% and 65% suppressed, respectively. MSM, MSW and women spent 86.9%, 82.6% and 82.8% of person-time on ART and 72.5%, 63.7% and 65% suppressed, respectively. Patients born in France (47%) and patients born abroad spent 87.9% and 81.9% of person-time on ART and 74.6% and 62.9% suppressed, respectively. Young men born abroad were found to spend the smallest person-time with non-detectable viral load (53% for MSW and 58.1% for MSM). Conclusions Despite free access to care and universal ART in France, disparities remain in the HIV continuum care across age, country of birth and way of HIV acquisition. Clinical and public health interventions targeting specific patients’ conditions are needed

Integrase strand transfer inhibitors and neuropsychiatric adverse events in a large prospective cohort

To analyse the frequency and causes of treatment discontinuation in patients who were treated with an integrase strand transfer inhibitor (INSTI), with a focus on neuropsychiatric adverse events (NPAEs)

Dolutegravir-based dual maintenance regimens combined with lamivudine/emtricitabine or rilpivirine: risk of virological failure in a real-life setting

International audienceBackground Maintenance ART with dolutegravir-based dual regimens have proved their efficacy among HIV-1-infected subjects in randomized trials. However, real-life data are scarce, with limited populations and follow-up. Objectives We assessed virological failure (VF) and resistance-associated mutations (RAMs) on dolutegravir maintenance regimens in combination with rilpivirine or with lamivudine or emtricitabine (xTC) and analysed the factors associated with VF. Methods Between 2014 and 2018, all HIV-1-infected adults included in the Dat’AIDS cohort and starting dolutegravir/rilpivirine or dolutegravir/xTC as a maintenance dolutegravir-based dual regimen were selected. VF was defined as two consecutive HIV RNA values >50 copies/mL or a single value >400 copies/mL. We compared cumulative genotypes before initiation of a maintenance dolutegravir-based dual regimen with genotype at VF. Results We analysed 1374 subjects (799 on dolutegravir/rilpivirine and 575 on dolutegravir/xTC) with a median follow-up of 20 months (IQR = 11–31) and 19 months (IQR = 11–31), respectively. VF occurred in 3.8% (n = 30) of dolutegravir/rilpivirine subjects and 2.6% (n = 15) of dolutegravir/xTC subjects. Among subjects receiving dolutegravir/rilpivirine, two genotypes harboured emerging RAMs at VF: E138K on NNRTI (n = 1); and E138K+K101E on NNRTI and N155H on INSTI (n = 1). Among subjects receiving dolutegravir/xTC, no new RAM was detected. The only predictive factor of VF on dolutegravir/rilpivirine was the history of failure on an NNRTI-based regimen (adjusted HR = 2.97, 95% CI = 1.28–6.93). No factor was associated with VF on dolutegravir/xTC. Conclusions In this large real-life cohort, dolutegravir/rilpivirine and dolutegravir/xTC sustained virological suppression and were associated with a low rate of VF and RAM emergence. Careful virological screening is essential before switching to dolutegravir/rilpivirine in virologically suppressed patients with a history of NNRTI therapy