Migrating to Long-Read Sequencing for Clinical Routine BCR-ABL1 TKI Resistance Mutation Screening

BackgroundHIV-1 resistance testing is recommended in clinical management and next-generation sequencing (NGS) methods are now available in many virology laboratories. ObjectivesTo evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing (Sequel, PacBio) for HIV-1 polymerase genotyping. Study design111 prospective clinical samples (83 plasma and 28 leukocyte-enriched blood fraction) were analyzed for routine HIV-1 resistance genotyping using Sanger sequencing, Vela NGS, and SMRT sequencing. We developed a SMRT sequencing protocol and a bio-informatics pipeline to infer antiretroviral resistance on both haplotype and variant calling approaches. ResultsThe polymerase was successfully sequenced by the three platforms in 98 % of plasma RNA samples for viral loads above 4 log copies/mL. The success rate decreased to 83 % using Sanger or Vela sequencing and to 67 % using SMRT sequencing for viral loads of 3 to 4 log copies/mL. Sensitivities of 50 %, 54 % and 61 % were obtained using SMRT, Vela, and Sanger sequencing, respectively, in cellular DNA from patients with prolonged undetectable plasma HIV-1 RNA. Ninety-eight percent of resistance-associated mutations (RAMs) identified with Sanger sequencing were detected using SMRT sequencing. Furthermore, 91 % of RAMs (> 5 % threshold) identified with Vela NGS were detected using SMRT sequencing. RAM quantification using Vela and SMRT sequencing was well correlated (Spearman correlation ρ = 0.82; P < 0.0001). ConclusionsSMRT sequencing of the full-length HIV-1 polymerase appeared performant for characterizing HIV-1 genotypic resistance on both RNA and DNA clinical samples. Long-read sequencing is a new tool for mutation haplotyping and resistance analysis.

To summarize the genetic characteristics of 671 Chinese pedigrees affected with Duchenne/Becker muscular dystrophy (DMD/BMD). Clinical data of the pedigrees were collected. Multiplex PCR, multiple ligation dependent probe amplification (MLPA), next generation sequencing (NGS), Sanger sequencing and long read sequencing were used to detect the variant of DMD gene in the probands and their mothers, and prenatal diagnosis was provided for high risk pregnant women. Among 178 pedigrees analyzed by multiplex PCR, 44 variants of the DMD gene were detected, with the genetic diagnosis attained in 110 pedigrees. Among 493 pedigrees analyzed by MLPA in combination with NGS or Sanger sequencing, 294 pathogenic/possible pathogenic variants were identified, among which 45 were unreported previously, and the genetic diagnosis attained in 484 pedigrees. Structural variants of the DMD gene were identified in two pedigrees by long-read sequencing. Among 444 probands, 341 have inherited the DMD gene variant from their mothers (76.8%). Among 390 women with a high-risk, 339 have opted to have natural pregnancy and 51 chose preimplantation genetic testing for monogenetic disease (PGT-M). The detection rate of neonatal patients and carriers following natural pregnancy was significantly higher than that for PGT-M. Combined application of MLPA, NGS, Sanger sequencing and long-read sequencing is an effective strategy to detect DMD/BMD. PGT-M can effectively reduce the risk of fetuses. Above finding has expanded the spectrum of DMD gene variants and provided a basis for reproductive intervention for pregnancies with a high risk for DMD/BMD.

BackgroundLong-read sequencing can be applied to generate very long contigs and even completely assembled genomes at relatively low cost and with minimal sample preparation. As a result, long-read sequencing platforms are becoming more popular. In this respect, the Oxford Nanopore Technologies–based long-read sequencing “nanopore" platform is becoming a widely used tool with a broad range of applications and end-users. However, the need to explore and manipulate the complex data generated by long-read sequencing platforms necessitates accompanying specialized bioinformatics platforms and tools to process the long-read data correctly. Importantly, such tools should additionally help democratize bioinformatics analysis by enabling easy access and ease-of-use solutions for researchers.ResultsThe Galaxy platform provides a user-friendly interface to computational command line–based tools, handles the software dependencies, and provides refined workflows. The users do not have to possess programming experience or extended computer skills. The interface enables researchers to perform powerful bioinformatics analysis, including the assembly and analysis of short- or long-read sequence data. The newly developed “NanoGalaxy" is a Galaxy-based toolkit for analysing long-read sequencing data, which is suitable for diverse applications, including de novo genome assembly from genomic, metagenomic, and plasmid sequence reads.ConclusionsA range of best-practice tools and workflows for long-read sequence genome assembly has been integrated into a NanoGalaxy platform to facilitate easy access and use of bioinformatics tools for researchers. NanoGalaxy is freely available at the European Galaxy server https://nanopore.usegalaxy.eu with supporting self-learning training material available at https://training.galaxyproject.org.

Objectives To explore the application of third-generation sequencing (TGS) for genetic diagnosis and prenatal genetic screening of thalassemia genes. Methods Two groups of subjects were enrolled in this study. The first group included 176 subjects with positive hematological phenotypes for thalassemia. Thalassemia-associated genes were detected simultaneously in each sample using both the PacBio TGS platform based on single-molecule real-time (SMRT) technology and the conventional PCR-reverse dot blot (PCR-RDB). Sanger sequencing was used for validation when results were discordant between the two methods. The second group included 53 couples with at least one partner having a positive thalassemia hematological phenotype, and they were screened for homotypic thalassemia variants by TGS, and the risk of pregnancies with babies presenting with severe thalassemia, was assessed. Results Of the 176 subjects, 175 had concordant genotypes between the two methods, including 63 normal subjects and 112 α- and/or β-thalassemia gene carriers, with a concordance rate of 99.43%. TGS detected a rare β-thalassemia gene variant −50 (G > A) that was not detected by conventional PCR-RDB. TGS identified seven of the 53 couples as homotypic thalassemia gene carriers, five of whom were at risk of pregnancies with severe thalassemia. Conclusion TGS could effectively detect common and rare thalassemia variants with high accuracy and efficiency. This approach would be suitable for prenatal thalassemia genetic screening in areas with high incidence of thalassemia.

Ultra Deep Sequencing (UDS) Allows More Sensitive Detection Of Tyrosine Kinase Inhibitor (TKI)-Resistant BCR-ABL Mutations That Would Influence Therapeutic Decision At The Time Of Switchover To Second- Or Third-Line Therapy

Tandem repeats (TRs) play essential roles in a variety of biological functions, and their abnormal expansions are significantly implicated in phenotypic variation and cause >60 human diseases. However, long TR regions cannot be reliably detected using short-read sequencing, and long-read sequencing enables accurate genome-wide detection of TRs. In recent years, various computational tools have been developed to detect and genotype TRs from long-read data. In this survey, we systematically categorize and review 39 computational tools designed for TR detection, visualization and functional interpretation. We discuss their strengths and limitations for TR detection from long-read sequencing data, highlighting current challenges and future directions to advance long-read TR detection methodologies.

Identification of a novel 10.3 kb deletion causing α0-thalassemia by third-generation sequencing: Pedigree analysis and genetic diagnosis

Transposable elements, such as Long INterspersed Elements (LINEs), are DNA sequences that can replicate within genomes. LINEs replicate using an RNA intermediate followed by reverse transcription and are typically a few kilobases in length. LINE activity creates genomic structural variants in human populations and leads to somatic alterations in cancer genomes. Long-read RNA sequencing technologies, including Oxford Nanopore and PacBio, can directly sequence relatively long transcripts, thus providing the opportunity to examine full-length LINE transcripts. This study focuses on the development of a new bioinformatics pipeline for the identification and quantification of active, full-length LINE transcripts in diverse human tissues and cell lines. In our pipeline, we utilized RepeatMasker to identify LINE-1 (L1) transcripts from long-read transcriptome data and incorporated several criteria, such as transcript start position, divergence, and length, to remove likely false positives. Comparisons between cancerous and normal cell lines, as well as human tissue samples, revealed elevated expression levels of young LINEs in cancer, particularly at intact L1 loci. By employing bioinformatics methodologies on long-read transcriptome data, this study demonstrates the landscape of L1 expression in tissues and cell lines.

Fabry disease (FD) exhibits a spectrum of clinical manifestations ranging from mild to severe, posing a diagnostic challenge, particularly in non-classic subtypes. Genetic testing remains a gold standard for a precise diagnosis of FD and is pivotal in genetic counseling. Although conventional approaches such as Sanger sequencing and short-read next-generation sequencing (NGS) have been successfully used to diagnose FD, they often fail to detect deep intronic variants, complex rearrangements, or large deletions or duplications. In contrast, long-read sequencing (LRS) enables comprehensive coverage of intronic and repetitive regions, facilitating precise identification of atypical variants missed by conventional methods. This case series reports two unrelated male patients with clinical, enzymatic, and pathological features consistent with FD, who tested negative for pathogenic variants in the alpha-galactosidase A (GLA) via Sanger sequencing and NGS. LRS identified novel non-coding variants in both patients. Patient 1 carried a ~1.7 kb insertion within intron 4, corresponding to part of a long interspersed nuclear element-1, while RNA sequencing revealed two new GLA transcripts. Patient 2 harbored a ~2.5 kb insertion within a SINE-VNTR-Alu retroposon element located in the 5′-untranslated region, with quantitative real-time PCR showing significantly reduced expression of normal GLA transcripts. These findings reveal non-coding variants that contribute to the missing heritability in FD, highlight this genomic region as a priority for future investigation, and demonstrate the potential utility of LRS in diagnostic workflows for unresolved FD cases.

Whole exome sequencing (WES) has become an increasingly common technique for identifying the genetic cause of Mendelian genetic diseases. However, it may fail to detect the complex regions of the genome. Here, we investigated the genetic etiology of a pedigree with autosomal dominant polycystic kidney disease (ADPKD) using a combination of WES, multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, and long-read sequencing (LRS). Initially, WES of the proband revealed a heterozygous variant c.7391G>C in PKD1 Exon 18, along with a heterozygous deletion of the 17th and 18th exons of PKD1 detected by exome-based copy number variation (CNV) analysis. MLPA confirmed the PKD1 heterozygous deletion of Exon 18. Except for c.7391G>C, Sanger sequencing identified four other heterozygous variants (c.7278T>C, c.7288C>T, c.7344C>G, and c.7365C>T) in Exon 18 of PKD1. Subsequently, LRS uncovered seven clustered substitution variants (c.7209+28C>T, c.7210-16C>T, c.7278T>C, c.7288C>T, c.7344C>G, c.7365C>T, and c.7391G>C), with six of them omitted by WES due to interference from PKD1 pseudogenes. Combining LRS results with cosegregation of the pedigree analysis, we found these variants were in cis and converted from PKD1 pseudogenes, covering a region of at least 282 bp. Notably, the paralogous sequence variants of c.7288C>T introduced a premature stop codon of PKD1, leading to a function loss, and were classified as pathogenic (PVS1+PS4+PM2) according to the ACMG/AMP guideline. Our study highlights the limitations of WES/MLPA and the importance of utilizing complementary tools like LRS for comprehensive variant detection in PKD1.

Notwithstanding the arrival of "third-generation sequencing," Sanger sequencing, developed in 1980, is still the most accurate and used method for sequencing, although on a smaller scale. It is a powerful resource for studying sequences and discovering polymorphisms and genes, as well as regulatory elements. There has already been described a wide range of possible problems with this very sensitive and accurate technology. Here, we show that a specific event, related to genomes rich in repetitive sequences, can mislead operators working with Sanger sequencing.

β-thalassemia is a common genetic disease mainly caused by point mutations in the β-globin gene, eliciting a high prevalence in South China. The aim of the present study is to identify a rare HBB: c.316-90A > G variant and provide the clinical and hematological features in two unrelated Chinese families. In this study, we collected eight subjects from two unrelated Chinese families. Conventional thalassemia gene testing was performed to investigate common α and β-thalassemia variants based on the PCR reverse dot hybridization technique. Third-generation sequencing (TGS) was utilized to examine the rare or novel HBA1, HBA2 and HBB gene variants, which will be further verified using Sanger sequencing. A rare HBB: c.316-90A > G variant was identified in the proband of Family 1 using TGS, and exhibited remarkably low levels of hemoglobin (Hb), Hb A2, MCV and MCH. The other members in Family 1 did not have the HBB: c.316-90A > G variant and elicited normal hematological screening results. In Family 2, the proband also carried the HBB: c.316-90A > G variant and exhibited low levels of MCV, MCH and Hb A2, but with normal Hb value. However, pedigree analysis results revealed that the proband's mother and nephew also carried the HBB: c.316-90A > G variant, but with normal hematological screening results. This study first conducts clinical and hematological analysis of the HBB: c.316-90A > G variant in two unrelated Chinese families, which provides valuable data for genetic counseling of the corresponding individuals.

We report a case of application of third-generation sequencing (TGS) combined with preimplantation genetic testing (PGT) for successful prevention of hereditary spastic paraplegia (HSP) caused by SPAST gene mutations and assess the value of PGT-M and TGS in managing hereditary spastic paraplegia. A family affected by HSP underwent whole exon sequencing (WES), and a c.1699G>T mutation in the SPAST gene was identified. The mutation site in the proband was confirmed through Sanger sequencing. A single nucleotide polymorphism (SNP) site flanking the SPAST gene mutation was selected as the genetic linkage marker, and a SNP haplotype carrying the mutated gene was constructed. Ovarian stimulation using an antagonist regimen was performed for oocyte retrieval, followed by intracytoplasmic sperm injection (ICSI) and embryo culture. Blastocyst trophectoderm cells were biopsied for preimplantation genetic testing for monogenic disorders (PGT-M) to allow the selection of disease-free embryos for transfer. In this cycle, a total of 20 oocytes were retrieved, among which 18 were successfully fertilized to result in 12 blastocysts eligible for biopsy. Genetic testing revealed that all the 12 blastocysts were successfully amplified and confirmed as euploidy. Among them, 8 blastocysts did not carry paternal mutations, and a high-quality euploid embryo was selected for frozen embryo transfer (FET). Subsequent amniotic fluid testing during pregnancy confirmed the absence of paternal mutations in the fetus, resulting in the birth of a healthy baby girl. For cases of genetic diseases with missing pedigree data, the application of third-generation sequencing and PGT-M technique can effectively block vertical transmission of SPAST gene mutation to the offspring, avoid pregnancy with an aneuploid embryo, and help families with autosomal dominant HSP obtain healthy offsprings.

Variable region analysis of 16S rRNA gene sequences is the most common tool in bacterial taxonomic studies. Although used for distinguishing bacterial species, its use remains limited due to the presence of variable copy numbers with sequence variation in the genomes. In this study, 16S rRNA gene sequences, obtained from completely assembled whole genome and Sanger electrophoresis sequencing of cloned PCR products from Serratia fonticola GS2, were compared. Sanger sequencing produced a combination of sequences from multiple copies of 16S rRNA genes. To determine whether the variant copies of 16S rRNA genes affected Sanger sequencing, two ratios (5:5 and 8:2) with different concentrations of cloned 16S rRNA genes were used; it was observed that the greater the number of copies with similar sequences the higher its chance of amplification. Effect of multiple copies for taxonomic classification of 16S rRNA gene sequences was investigated using the strain GS2 as a model. 16S rRNA copies with the maximum variation had 99.42% minimum pairwise similarity and this did not have an effect on species identification. Thus, PCR products from genomes containing variable 16S rRNA gene copies can provide sufficient information for species identification except from species which have high similarity of sequences in their 16S rRNA gene copies like the case of Bacillus thuringiensis and Bacillus cereus. In silico analysis of 1,616 bacterial genomes from long-read sequencing was also done. The average minimum pairwise similarity for each phylum was reported with their average genome size and average “unique copies” of 16S rRNA genes and we found that the phyla Proteobacteria and Firmicutes showed the highest amount of variation in their copies of their 16S rRNA genes. Overall, our results shed light on how the variations in the multiple copies of the 16S rRNA genes of bacteria can aid in appropriate species identification.

SummaryDespite the continuous discovery of new transcript isoforms, fueled by the recent increase in accessibility and accuracy of long-read RNA sequencing data, functional differences between isoforms originating from the same gene often remain obscure. To address this issue and enable researchers to assess potential functional consequences of transcript isoform variation on the proteome, we developed IsoTV. IsoTV is a versatile pipeline to process, predict and visualize the functional features of translated transcript isoforms. Attributes such as gene and isoform expression, transcript composition and functional features are summarized in an easy-to-interpret visualization. IsoTV is able to analyze a variety of data types from all eukaryotic organisms, including short- and long-read RNA-seq data. Using Oxford Nanopore long read data, we demonstrate that IsoTV facilitates the understanding of potential protein isoform function in different cancer cell types.Availability and implementationIsoTV is available at https://github.molgen.mpg.de/MayerGroup/IsoTV, with the corresponding documentation at https://isotv.readthedocs.io/.Supplementary informationSupplementary data are available at Bioinformatics online.