scholarly journals HyINDEL – A Hybrid approach for Detection of Insertions and Deletions

2021 ◽  
Author(s):  
Alok Thatikunta ◽  
Nita Parekh

Insertion and deletion (INDELs) mutations, the most common type of structural variation in the human genome, have been implicated in numerous human traits and diseases including rare genetic disorders and cancer. Next generation sequencing (NGS) technologies have drastically reduced the cost of sequencing whole genomes, greatly contributing to genome-wide detection of structural variants. However, due to large variations in INDEL sizes and presence of low complexity and repeat regions, their detection remains a challenge. Here we present a hybrid approach, HyINDEL, which integrates clustering, split-mapping and assembly-based approaches, for the detection of INDELs of all sizes (from small to large) and also identifies the insertion sequences. The method starts with identifying clusters of discordant and soft-clip reads which are validated by depth-of-coverage and alignment of soft-clip reads to identify candidate INDELs, while the assembly -based approach is used in identifying the insertion sequence. Performance of HyINDEL is evaluated on both simulated and real datasets and compared with state-of-the-art tools. A significant improvement in recall and F-score metrics as well as in breakpoint support is observed on using soft-clip alignments. It is freely available at https://github.com/alok123t/HyINDEL.

2017 ◽  
Author(s):  
Claire Marchal ◽  
Takayo Sasaki ◽  
Daniel Vera ◽  
Korey Wilson ◽  
Jiao Sima ◽  
...  

ABSTRACTCycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease. Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyze genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing RT allelic asynchrony, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.


Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 92 ◽  
Author(s):  
Shannon J. McKie ◽  
Anthony Maxwell ◽  
Keir C. Neuman

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.


Diagnostics ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 230 ◽  
Author(s):  
Muzammil H. Syed ◽  
Abdelrahman Zamzam ◽  
Jason Valencia ◽  
Hamzah Khan ◽  
Shubha Jain ◽  
...  

Chronic limb-threatening ischemia (CLTI) results in devastating complications such as lower-limb amputations. In this study, a genome-wide plasma microRNAs (miRNA) sequencing was performed to identify miRNA(s) associated with CLTI. Blood samples were collected from early stage CLTI patients (ABI < 0.9) and non-PAD controls (ABI ≥ 0.9) for 3 experiments: discovery phase (n = 23), confirmatory phase (n = 52) and validation phase (n = 20). In the discovery phase, next generation sequencing (NGS) was used to identify miRNA circulating in the plasma CLTI (n = 13) patients, compared to non-PAD controls (n = 10). Two down-regulated miRNAs (miRNA-6843-3p and miRNA-6766-5p) and three upregulated miRNAs (miRNA-1827, miRNA-320 and miRNA-98-3p) were identified (≥2-fold change). In the confirmatory phase, these 5 deregulated miRNAs were further investigated in non-PAD (n = 21) and CTLI (n = 31) patients using qRT-PCR. Only miRNA-1827 was found to be significantly upregulated (≥3-fold, p-value < 0. 001) in the CLTI group. Lastly, to minimize the influence of confounding factors, miRNA-1827 plasma levels were validated in a third cohort of CLTI patients (n = 10) matched to non-PAD controls (n = 10). Our analysis demonstrated that miRNA-1827 expression was increased in the CLTI cohort (≥2-folds, p-value < 0.001). In summary, circulating miRNA-1827 is significantly elevated in patients with CLTI.


2015 ◽  
Vol 97 ◽  
Author(s):  
TONY SHEN ◽  
ARIEL LEE ◽  
CAROL SHEN ◽  
C.JIMMY LIN

SummaryThere are an estimated 6000–8000 rare Mendelian diseases that collectively affect 30 million individuals in the United States. The low incidence and prevalence of these diseases present significant challenges to improving diagnostics and treatments. Next-generation sequencing (NGS) technologies have revolutionized research of rare diseases. This article will first comment on the effectiveness of NGS through the lens of long-tailed economics. We then provide an overview of recent developments and challenges of NGS-based research on rare diseases. As the quality of NGS studies improve and the cost of sequencing decreases, NGS will continue to make a significant impact on the study of rare diseases moving forward.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3266-3266
Author(s):  
Cécile Bally ◽  
Aline Renneville ◽  
Lionel Adès ◽  
Claude Preudhomme ◽  
Hugues de Thé ◽  
...  

Abstract Background TP53 mutations inactivating p53 protein, often associated with loss of the remaining TP53 allele through 17p deletion, are major prognostic factors in many hematological malignancies, including CLL, myeloma, AML and MDS. In AML and MDS, they are usually associated with complex karyotype (including del 17p) and very poor prognosis (Blood 1991, 78(7):1652-7 , Bejar, NEJM 2011), including after allogeneic SCT (Middeke JM, Blood 2014) but they are also seen in lower risk MDS with isolated del 5q, where they confer resistance to Lenalidomide (Jadersten, JCO 2011). The advent of Next Generation Sequencing (NGS) techniques has improved the detection of such mutations, by allowing the identification of small mutated clones. Other detection methods may prove interesting, especially functional methods like FASAY ( Functional Assay of Separated Allele in Yeast) , an easy and sensitive method that detects TP53 mutations by assessing the p53 function as transcription factor (Flaman et al, PNAS 1995). We compared the detection of TP53 mutations in MDS and AML by FASAY and NGS approaches. Methods The 84 patients analyzed included 10 AML, 10 higher risk MDS, and 64 lower risk MDS with del 5q. RNA and DNA were extracted from marrow mononuclear cells. TP53 mutations were detected on RNA by FASAY where, after amplification of the TP53 mRNA, the PCR product is co transfected with an open gap repair plasmid leading, by homologous recombination, to p53 protein expression in the yeast. The yeast strain used is dependent on p53 functionality for growth and color and detection of more than 10% of small red yeast colonies defines a non-functional FASAY result. All non-functional FASAY were confirmed by the split versions of the test and TP53 defects were characterized by Sanger sequencing. The detection limit is around 10% in our hands (Manie E, Cancer Res 2009). In parallel, TP53 mutations were detected on DNA by NGS using the IRON II plate design and pyrosequencing on a GS Junior System (Roche). (Kohlmann, Leukemia 2011).FASAY (+Sanger sequencing) and NGS were performed in two different labs. Results By FASAY, 47 patients (56%) had a functional p53 and 37 cases (44%) a non-functional p53 and a mutation was confirmed by Sanger in all non functional cases. By NGS analysis, no TP53 mutation was found in 47 cases (56%) and a mutation was detected in 37 cases (44%). In the 37 mutated cases by NGS, the median proportion of mutated allele was 35% (range 3 to 99%), including a median of 72%, 35%, 25 % in AML, higher risk MDS and lower risk MDS with del 5q, respectively. The mutated clone size was lower than 10% in only 2 patients who both had lower risk MDS with del 5q (3 and 6%, respectively). A perfect correlation between FASAY and NGS was found in 80 (95.5%) cases. The 4 discordant cases included a mutation detected only by FASAY in 2 cases, and only by NGS in 2 cases. Undetected mutations by NGS were insertions of intronic sequences (intron 9) not explored by the technique used. These insertions resulted in non-functional protein well detected by FASAY which analyses the global cDNA sequence including splicing defects. Undetected mutations by FASAY were mutations in which the percentage of mutated alleles was less than 10% (3% and 6 % respectively). Finally, while the cost of NGS analysis for TP53 mutation is around 200 euros when performed alone (and around 2000 euros when combined to analysis of the 30 main other genes involved in MDS and AML), the cost of the FASAY technique is around 20 euros (prices including reagents only). Conclusion The FASAY technique is a cheap method, that in spite of a sensitivity of only 10%, was able to detect 98% of TP53 mutations detected by NGS. In fact those mutations appear to involve generally relatively large clones in MDS and AML. FASAY could also detect 2 atypical intronic mutations overlooked by NGS. Demonstrating in such difficult cases that the resulting p53 protein is non functional and therefore probably has pathophysiological significance, is an advantage of FASAY .The combination of the 2 methods, and especially the combination of DNA and RNA analysis, may be useful in such cases. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Vol 7 (8) ◽  
pp. 636
Author(s):  
Chi-Ching Tsang ◽  
Jade L. L. Teng ◽  
Susanna K. P. Lau ◽  
Patrick C. Y. Woo

Next-generation sequencing (NGS) technologies have recently developed beyond the research realm and started to mature into clinical applications. Here, we review the current use of NGS for laboratory diagnosis of fungal infections. Since the first reported case in 2014, >300 cases of fungal infections diagnosed by NGS were described. Pneumocystis jirovecii is the predominant fungus reported, constituting ~25% of the fungi detected. In ~12.5% of the cases, more than one fungus was detected by NGS. For P. jirovecii infections diagnosed by NGS, all 91 patients suffered from pneumonia and only 1 was HIV-positive. This is very different from the general epidemiology of P. jirovecii infections, of which HIV infection is the most important risk factor. The epidemiology of Talaromyces marneffei infection diagnosed by NGS is also different from its general epidemiology, in that only 3/11 patients were HIV-positive. The major advantage of using NGS for laboratory diagnosis is that it can pick up all pathogens, particularly when initial microbiological investigations are unfruitful. When the cost of NGS is further reduced, expertise more widely available and other obstacles overcome, NGS would be a useful tool for laboratory diagnosis of fungal infections, particularly for difficult-to-grow fungi and cases with low fungal loads.


2020 ◽  
Vol 9 (8) ◽  
pp. 2633 ◽  
Author(s):  
Alain Calender ◽  
Thomas Weichhart ◽  
Dominique Valeyre ◽  
Yves Pacheco

Sarcoidosis is a complex disease that belongs to the vast group of autoinflammatory disorders, but the etiological mechanisms of which are not known. At the crosstalk of environmental, infectious, and genetic factors, sarcoidosis is a multifactorial disease that requires a multidisciplinary approach for which genetic research, in particular, next generation sequencing (NGS) tools, has made it possible to identify new pathways and propose mechanistic hypotheses. Codified treatments for the disease cannot always respond to the most progressive forms and the identification of new genetic and metabolic tracks is a challenge for the future management of the most severe patients. Here, we review the current knowledge regarding the genes identified by both genome wide association studies (GWAS) and whole exome sequencing (WES), as well the connection of these pathways with the current research on sarcoidosis immune-related disorders.


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