NGS-based Method Research Articles

Detection of leukemia gene fusions by targeted RNA-sequencing in routine diagnostics

BackgroundWe have evaluated an NGS-based method to detect recurrent gene fusions of diagnostic and prognostic importance in hematological malignancies. Our goal was to achieve a highly specific assay with a simple workflow, short turnaround time and low cost.MethodThe assay uses a commercially available anchored multiplex PCR panel for target enrichment and library preparation, followed by sequencing using a MiSeq instrument. The panel includes all recurrent gene fusions in AML and ALL and is designed to detect gene-specific fusions without prior knowledge of the partner sequence or specific break points. Diagnostic RNA samples from 27 cases with hematological malignancies encompassing 23 different transcript variants were analyzed. In addition, 12 cases from a validation cohort were assessed.ResultAll known fusion transcripts were identified with a high degree of confidence, with a large number of reads covering the breakpoints. Importantly, we could identify gene fusions where conventional methods had failed due to cryptic rearrangements or rare fusion partners. The newly-identified fusion partners were verified by RT-PCR and transcript-specific qPCR was designed for patient-specific follow-up. In addition, 12 cases were correctly assessed in a blind test, without prior knowledge of molecular cytogenetics or diagnosis.ConclusionIn summary, our results demonstrate that targeted RNA sequencing using anchored multiplex PCR can be implemented in a clinical laboratory for the detection of recurrent and rare gene fusions in hematological diagnostic samples.

Identification of Novel Alleles and Structural Haplotypes of Major Histocompatibility Complex Class I and DRB Genes in Domestic Cat (Felis catus) by a Newly Developed NGS-Based Genotyping Method.

The major histocompatibility complex (MHC) is a highly polymorphic and duplicated genomic region that encodes transplantation and immune regulatory molecules. Although it is well-known that particular MHC allelic polymorphisms and haplotypes are genetically relate to immune-mediated diseases detailed information of the cat MHC (Feline Leukocyte Antigen; FLA) genetic and haplotypic structure and diversity is limited in comparison to humans and many other species. In this study, to better understand the degree and types of allele and allelic haplotype diversity of FLA-class I (FLA-I) and FLA-DRB loci in domestic cats, we identified six expressible FLA-I loci in peripheral white blood cells by in silico estimation of the coding exons and NGS-based amplicon sequencing using five unrelated cats. We then used a newly developed NGS-based genotyping method to genotype and annotate 32 FLA-I and 16 FLA-DRB sequences in two families of 20 domestic cats. A total of 14 FLA-I and seven FLA-DRB were identified as novel polymorphic sequences. Phylogenetic analyses grouped the sequences into six FLA-I (FLA-E/H/K, FLA-A, FLA-J, FLA-L, FLA-O and a tentatively named FLA-E/H/K_Rec) and four FLA-DRB (FLA-DRB1, FLA-DRB3, FLA-DRB4, and FLA-DRB5) lineages. Pedigree analysis of two cat families revealed eight distinct FLA structural haplotypes (Class I – DRB) with five to eight FLA-I and two to three FLA-DRB transcribed loci per haplotype. It is evident that the eight FLA haplotypes were generated by gene duplications and deletions, and rearrangements by genetic recombination with the accumulation and/or inheritance of novel polymorphisms. These findings are useful for further genetic diversity analysis and disease association studies among cat breeds and in veterinary medicine.

Abstract B62: Evaluation of systemic RNA-based cancer vaccine induced T-cell responses via mouse T-cell receptor (TCR) profiling

Abstract Identification of biomarkers is pivotal towards the proper development of cancer immunotherapies, in particular for designing the most effective regimens and identifying patient subgroups most likely to benefit from a given treatment. T-cell receptor (TCR) repertoire emerged as a promising biomarker in this respect, as it allows measurement of the dynamics of the T cell response both in time and in different compartments. In this work, we developed and evaluated an NGS-based mouse TCR profiling method (TCR alpha and TCR beta) for discovery and validation of biomarkers related to the antitumoral T-cell response in different compartments upon systemic administration of an RNA-based cancer vaccine (RNA-lipoplex). The in-house established mouse TCR profiling protocol was able to detect blood-spiked known-TCR sequences down to a frequency of 0,001% spiked cells for both TCR alpha and TCR beta chains and the relative frequencies of detected TCRs in the tumor correlated well with the degree of T-cell infiltration into the same tumors assessed by flow cytometry. Comparative analysis of changes in the TCR repertoire between blood and tumor upon repetitive vaccination with RNA-lipoplexes encoding immunodominant epitope of chicken ovalbumin (OVA257-264) revealed that richness as well as diversity of endogenous TCRs were generally lower in B16-OVA tumors compared to blood and decreased with further vaccination. Interestingly, top clones overlapped increasingly in blood and tumor until after the second vaccination, but drifted apart after the third vaccination, with the TCR repertoire in the tumor narrowing down. This overlap was less evident when mice were inoculated with B16-F10 tumors. Top clones within the same compartment were found to be highly variable between individual animals, complicating comparative analyses. Monitoring intra-individual TCR repertoire changes in the blood of a given mouse over time, however, indicated a tendency for decreasing richness and diversity following a single RNA vaccination, which may be pointing at repertoire adaptation and antigen focus. Supporting this notion, tumors isolated at late time-points exhibited a lower diversity than those investigated at earlier time-points and at baseline. In contrast to RNA vaccination, richness and diversity were reduced in response to treatment with an agonistic anti-CD40 antibody in blood and spleen but were similar among tumors. Consequently, richness and diversity in the periphery may not necessarily be predictive of corresponding repertoire changes in the tumor microenvironment. Citation Format: Lena Mareen Kranz, Barbara Schrörs, Thomas Bukur, Christian Albrecht, Lorieta Leppin, Martin Suchan, Marta Faryna, Eileen Powalsky, Rainer Hipfel, Özlem Türeci, Ugur Sahin, Mustafa Diken. Evaluation of systemic RNA-based cancer vaccine induced T-cell responses via mouse T-cell receptor (TCR) profiling [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B62.

MCSeEd (Methylation Context Sensitive Enzyme ddRAD): A New Method to Analyze DNA Methylation.

Methylation context sensitive enzyme ddRAD (MCSeEd) is a NGS-based method for genome-wide investigations of DNA methylation at different contexts requiring only low to moderate sequencing depth. It is particularly useful for identifying methylation changes in experimental systems challenged by biotic or abiotic stresses or at different developmental stages.

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S\u201323S rRNA region

BackgroundMany members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S–23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S–23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S–23S rRNA region.MethodsThirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S–23S rRNA region.ResultsThis study allowed obtainment and deposition of reference sequences of the 16S–23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S–23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level.ConclusionsBased on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S–23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S–23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

Molecular Characteristics and Disease Burden Metrics Determined By Next-Generation Sequencing on Circulating Tumor DNA Correlate with Progression Free Survival in Previously Untreated Diffuse Large B-Cell Lymphoma

Introduction: Due to the range of biological and molecular heterogeneity in diffuse large B-cell lymphoma (DLBCL), personalized risk stratification and treatment is a promising avenue to improving outcomes. Although most risk stratification depends primarily on clinical data (e.g. IPI), the addition of molecular, genomic or disease burden (e.g. quantitative PET imaging) features in DLBCL could help better stratify patients (pts) according to disease biology or burden. Such data are often hard to obtain in routine clinical settings; current methods remain limited by the need for tissue samples and low reproducibility in daily practice. A single method to assess such molecular markers from plasma samples could enable a standardized process. Here, we use a circulating tumor DNA (ctDNA)-based next-generation sequencing (NGS) method on pre-treatment plasma samples from first-line DLBCL pts to show prognostic correlations from molecular and disease burden assessments. Methods: We performed targeted NGS on plasma samples from 310 previously untreated DLBCL pts enrolled in the GOYA study (NCT01287741) with a custom DLBCL-specific panel using a workflow optimized for ctDNA. Cell-free DNA (cfDNA) was isolated from plasma and an average of 30.7ng (range, 0.7-50ng) of cfDNA was used. Library preparation and NGS were performed with a modified AVENIO ctDNA workflow, with a custom panel of ~314kb designed to cover regions relevant for cell-of-origin (COO) and minimal residual disease in DLBCL. Single nucleotide variants (SNVs), insertions/deletions (indels), and fusions were determined, and criteria based on publicly available data removed non-tumor specific variants. For each sample, number of tumor genome copies per mL of plasma (MMPM), a measure of tumor burden, was calculated using the allele fractions of variant calls and cfDNA mass. Variant calls from 230 pts were used to build a machine learning model to determine COO, which was tested on the remaining 80 pts. Variant and COO calls were correlated with data from corresponding tissue samples, including mutations from FoundationOne® Heme and gene expression-based COO determination from Lymph2Cx (NanoString). Variant, COO, and disease burden assessments from ctDNA NGS were also compared with clinical variables, including IPI, baseline quantitative PET imaging, and progression-free survival (PFS) data. Results: 77% of SNVs (958/1247), 63% of indels (72/115), and 74% of fusions (49/66) detected with the FoundationOne assay were detected in the plasma samples. COO calls from plasma showed 93% concordance with Lymph2Cx calls from tissue when both methods classified a sample, with 37/40 germinal center B-cell (GCB) agreeing and 17/18 non-GCB agreeing in the test set. Subsequently, correlation of various metrics from the ctDNA assay with clinical outcomes was assessed. Non-GCB pts showed a trend towards worse PFS when compared with GCB pts (hazard ratio [HR], 1.23; 95th percentile: 0.79─1.92; p=0.32). Additionally, worse PFS was observed for pts with MYC fusions (n=12; HR, 2.83; 95th percentile: 1.29─6.21; p=0.010) and TP53 SNVs or indels (n=99; HR, 1.75; 95th percentile: 1.13─2.70; p=0.031), with a similar trend for BCL2 fusions (n=31; HR, 1.96; 95th percentile: 0.94─4.07; p=0.072). Baseline MMPM was significantly correlated with total metabolic tumor volume (TMTV) as measured by PET (r=0.36; p<0.001) and sum of the product of diameters (SPD; r=0.13; p=0.022). Higher MMPM values corresponded to higher IPI scores (ANOVA p<0.001), higher likelihood of bulky disease (p=0.019), and worse PFS as a continuous variable (HR, 1.46; p=0.0006; Figure 1). In a multivariate model accounting for TMTV, MMPM was not prognostic (p=0.15), but in a model containing SPD and MMPM, both were independently prognostic (MMPM p=0.010; SPD p=0.0046), suggesting that SPD and MMPM provide complementary prognostic information. Interestingly, in this sample set, activated B-cell samples also showed a significantly higher MMPM than GCB and unclassified samples (p=0.0017). Even when considering COO and IPI in a multivariate analysis, MMPM remained correlated with PFS (HR, 1.23; p=0.079). Conclusions: We describe a single NGS-based method, which calls variants, determines COO, and assesses tumor burden from plasma. Using these results, we show that pre-treatment plasma-based molecular and tumor burden measurements in previously untreated DLBCL pts correlate with PFS. Disclosures Tabari: F. Hoffmann-La Roche: Equity Ownership; Roche Sequencing Solutions: Employment. Lovejoy:Roche Sequencing Solutions: Employment. Lin:Roche Sequencing Solutions: Employment; Veracyte: Other: Veracyte (spouse). Bolen:F. Hoffmann-La Roche: Equity Ownership; Genentech, Inc.: Employment. Saelee:Roche Sequencing Solutions: Employment. Lefkowitz:Roche Sequencing Solutions: Employment. Kurtz:Roche: Consultancy. Vitazka:Roche Sequencing Solutions: Employment. Venstrom:F. Hoffmann-La Roche Ltd: Employment. Nielsen:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Parreira:F.Hoffmann-La Roche Ltd: Employment, Equity Ownership, Honoraria. Klass:Roche Sequencing Solutions: Employment; Roche: Equity Ownership. Luong:Roche Sequencing Solutions: Employment.

Detection of neoantigen-reactive T cell clones based on the clonal expansion using next-generation sequencing of T cell receptor β complementarity-determining region 3

Development of mechanism-driven biomarkers for immune checkpoint inhibitors (ICIs) in cancer immunotherapy requires sensitive and efficacious assays to identify tumor antigen (Ag)-specific T cells. We demonstrated the concept for a sensitive method to determine Ag-reactive T cell clones based on clonal expansion using model neoantigens (NeoAgs) rather than cytokine production. Sequential increase in T cell clonal frequencies following Ag stimulation was detected by next generation sequencing (NGS) of T cell receptor β (TCR β) complementarity-determining region 3 (CDR3), with a higher sensitivity than that of enzyme-linked immunospot (ELISPOT) by 100-fold. The TCRβ CDR3 sequences could represent useful markers to track dynamic changes during immunotherapy. The TCRβ NGS-based method could represent a novel platform both for the development of new biomarkers as well as several therapeutic options.

7. GENETIC TESTING FOR COPY NUMBER VARIATIONS IN FIRST POLAR BODIES AND OOCYTES BY SINGLE-CELL NEXT-GENERATION SEQUENCING

Introduction Female meiotic errors are the main cause of whole chromosomal aneuploidy in human embryos and significantly contribute to early embryo loss. Meiotic chromosomal malsegregation occurs during divisions of the maturing oocyte in oogenesis concurrently with extrusion of two polar bodies: polar body first (PB1) and polar body second (PB2). Based on meiosis mechanism, genome of a single oocyte can be deduced by analyzing its sibling polar bodies. PB genetic testing can identify abnormalities of maternal meiotic origin and may be the only form of preimplantation genetic testing for aneuploidies (PGT-A) accepted by patients who object embryo biopsy for ideological reasons. In the majority of previous studies, array comparative genomic hybridization (aCGH) was a method of choice for comprehensive chromosome testing in polar bodies. Here, we test the use of single-cell next-generation sequencing (NGS) method in polar body testing for detecting copy number variations (CNVs) in PB1 genome and validate methods for whole genome amplification (WGA) of PB1. Material and methods In this study, single-cell NGS technology was used to asses ploidy status of oocytes and their sibling PB1. A total of 120 single cells (60 first polar bodies and 60 oocytes) were collected from in vitro fertilization (IVF) couples and subjected for WGA using four different kits based on three distinct amplification systems: (i) polymerase chain reaction (PCR-based WGA), (ii) multiple displacement amplification (MDA) or (iii) multiple annealing and looping-based amplification cycles (MALBAC). WGA products were processed and analyzed with VeriSeq PGS MiSeq kit on MiSeq sequencing platform (Illumina) and random samples were validated with the aCGH method. Amplification uniformity and accuracy for CNV detection of the four tested kits were evaluated. Results Ploidy status was determined for 112 of 120 (93,3%) samples (84/112 based on direct analysis and 28/112 based on indirect analysis of the well-amplified sibling PB1 or oocyte). Estimated aneuploidy rate for the studied group which resembles the total oocyte abnormality resulting from meiosis I errors rate was 33% (37/112 samples). In the group of well-amplified PB1-oocyte pairs (33/60) a 97% concordance between the chromosomal status of PB1 and the corresponding oocyte was observed. Both whole chromosome nondisjunction (6 of 37 aneuploid samples) and abnormal chromatid pre-division and segregation were detected (31 of 37 aneuploid samples). The result is concordant with previous studies - abnormal chromatid segregation was the dominant aneuploidy-causing mechanism. Differences in WGA kits performance including amplification uniformity, aneuploidy calling potential and a number of interpretable NGS results were evaluated and the most optimal kit for PB1 genome WGA was pre-selected. Results from aCGH validation of the randomly selected samples showed full consistency with the NGS results. Conclusions The NGS-based method used for PB1 genome analysis showed a high predictive potential of PB1 in deducing ploidy status of the corresponding oocyte and is a promising method for PGT-A of oocytes. The study also contributes to a better understanding of the chromosome segregation patterns and maternal meiosis errors. Supported by: POIR.02.03.02-14- 0092/17

Next-generation sequencing-based bulked segregant analysis for QTL mapping in the heterozygous species Brassica rapa.

An improved protocol of QTL-seq, an NGS-based method for bulked segregant analysis we previously developed in rice, allowed successful mapping of QTLs of interest in the highly heterozygous genome of B. rapa, demonstrating the power of this elegant method for genetic analyses in heterozygous species of economic importance. Recent advances in next-generation sequencing (NGS) and the various NGS-based methods developed for rapidly identifying candidate genes of interest have accelerated genetic analysis mainly in the model plants rice and Arabidopsis. Brassica rapa includes several economically important crops such as Chinese cabbage, turnip and various leafy vegetables. The application of NGS-based approaches for the analysis of B. rapa has been limited mainly due to its highly heterozygous genome and poor quality of the reference genome sequence currently available for this species. In this study, we have improved QTL-seq, a method for NGS-based bulked segregant analysis we previously developed in rice, extending its applicability for accelerating the genetic analysis and molecular breeding of B. rapa. Addition of new filters to the original QTL-seq pipeline allowed removal of spurious single-nucleotide polymorphisms caused by alignment/sequencing errors and variability between parents, significantly improving accuracy of the analysis. As proof of principle, we successfully applied the new approach to identify candidate genomic regions controlling flowering and trichome formation using segregating F2 progeny obtained from crosses made between cultivars of B. rapa showing contrasting phenotypes for these traits. We strongly believe that the improved QTL-seq method reported here will extend the applicability of NGS-based genetic analysis not only to B. rapa but also to other plant species of economic importance with heterozygous genomes.

Abstract 3492: A targeted semi-conductor based next-generation sequencing (NGS) test to characterize microsatellite instability in FFPE tumor samples

Abstract Microsatellite instability (MSI) arises from defects in the mismatch repair (MMR) system and is associated with hypermutability of short DNA sequence repeats. Defects in MMR are observed in diverse cancer types but are common in colorectal, gastric and endometrial cancers. MSI testing is increasingly important for patient management. For example, in diverse cancer types, MSI is associated with favorable response to immune checkpoint inhibitors. The mainstay of MSI testing has been PCR/fragment analysis or IHC to monitor loss of expression of MMR proteins. Although generally robust, these methods are single biomarker tests that may consume limited biopsy samples. For this reason, MSI testing has been recently incorporated into NGS tests. However, targeted amplicon based NGS tests for MSI that consume limited sample input and provide fast turn-around time remain an unmet need. Herein, we describe a targeted NGS-based method to assess MSI that leverages Ion AmpliSeq™ or Ion AmpliSeq™ HD multiplex PCR and Ion GeneStudio™ S5 next-generation sequencing. The test is comprised of diverse microsatellite markers including mono- and di-nucleotide repeats that range from 10 to 40 bp. A novel algorithm was developed that leverages the unique signal processing properties inherent in semi-conductor sequencing and workflows were developed for tumor only samples as well as paired tumor-normal samples. The test provides results for individual microsatellites and generates an MSI score for the sample. The performance of the assay was verified over a large cohort of colorectal, gastric and endometrial cancer samples with MSI status independently assigned by orthogonal on-market tests. The MSI panel can be used by itself or integrated into larger targeted sequencing panels. MSI tests that leverage the inherent advantages of targeted semiconductor sequencing, low sample input and fast turn-around time, will support expanded research opportunities into the association of MSI with other targeted alterations and help elucidate the interaction of MSI, DNA repair defects, and the response to immune checkpoint inhibition. Citation Format: Anelia Kraltcheva, Sameh El-Difrawy, Asha Kamat, Janice Au-Young, Simon Cawley, Seth Sadis. A targeted semi-conductor based next-generation sequencing (NGS) test to characterize microsatellite instability in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3492.

Assessment and characterization of the bacterial community structure in advanced activated sludge systems

The present study is aimed to assess and characterize the structure of bacterial community in advanced activated sludge systems. In particular, activated sludge samples were collected from an Integrated Fixed-film Activated Sludge – Membrane Bioreactor pilot plant under a University of Cape Town configuration with in-series anaerobic (Noair)/anoxic (Anox)/aerobic (Oxy) reactors – and further analyzed. The achieved results – based on Next Generation Sequencing (NGS) of 16S rDNA amplicons – revealed that the bacterial biofilm (bf) communities on plastic carriers of Oxy and Anox reactors had a greater diversity compared to suspended (sp) bacterial flocs of Oxy, Anox and Noair. Indeed, the Shannon diversity indices of both biofilm communities were higher than those of suspended growth samples (Oxy-bf = 4.1 and Anox-bf = 4.2 vs. Oxy-sp = 3.4, Anox-sp = 3.5 and Noair-sp = 3.4). The most striking differences have been reported in Rhodobacteraceae being more abundant in biofilm specimens than in suspended biomass samples. The vast majority of the identified bacteria differs from those obtained by culture dependent method, thus suggesting that NGS-based method is really suitable to analyze the bacterial community composition, even in advanced systems for wastewater treatment.

DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes

PurposeGross duplications are ambiguous in terms of clinical interpretation due to the limitations of the detection methods that cannot infer their context, namely, whether they occur in tandem or are duplicated and inserted elsewhere in the genome. We investigated the proportion of gross duplications occurring in tandem in breast cancer predisposition genes with the intent of informing their classifications. MethodsThe DNA breakpoint assay (DBA) is a custom, paired-end, next-generation sequencing (NGS) method designed to capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2. ResultsDBA allowed us to ascertain breakpoints for 44 unique gross duplications from 147 probands. We determined that the duplications occurred in tandem in 114 (78%) carriers from this cohort, while the remainder have unknown tandem status. Among the tandem gross duplications that were eligible for reclassification, 95% of them were upgraded to pathogenic. ConclusionDBA is a novel, high-throughput, NGS-based method that informs the tandem status, and thereby the classification of, gross duplications. This method revealed that most gross duplications in the investigated genes occurred in tandem and resulted in a pathogenic classification, which helps to secure the necessary treatment options for their carriers.

Next generation sequencing as the new gold standard for minimal residual disease detection in B-ALL

Cheng et al . (1) in their study explored the use of the next-generation sequencing (NGS) technology to evaluate post-remission minimal residual disease (MRD) in B acute lymphoblastic leukaemia/lymphoma (B-ALL) patients and its potential to be implemented in clinical settings. The authors propose an NGS-based method with an applicability of around 90% of B-ALL patients and a sensitivity of up to 10−6 and losing only 6.25% of the cases where leukaemia-specific clonotypes were not identified.

Analysis of microsatellite loci in tree of heaven (Ailanthus altissima (Mill.) Swingle) using SSR-GBS

Microsatellite markers are still the marker of choice for many research questions in the field of forest genetics. However, the number of available markers is often low for species that have not been studied intensively like the tree of heaven (Ailanthus altissima). During the last decade, next-generation sequencing (NGS) has offered advanced techniques for efficiently identifying microsatellite markers and accurately genotyping samples. Here, we identify new microsatellite markers for the tree of heaven by applying an NGS-based method using the Illumina MiSeq platform. NGS technology was proved to be an effective method for fast and cost-efficient identification of microsatellite markers by implementing a genotyping-by-sequencing approach based on Illumina amplicon sequencing (SSR-GBS). We screened three populations from Eastern Austria for genetic variation at 19 newly identified microsatellite loci. We tested two different genotyping approaches: (1) considering only allele lengths (forming a so-called “allele length dataset”), (2) taking also single nucleotide polymorphisms (SNPs) within the amplified fragments into account (forming a so-called “SNP dataset”). The results revealed higher values for all genetic diversity parameters, as well as a better resolution of genetic assignment, when the latter approach was followed. Thus, by taking advantage of sequence information which is provided by SSR-GBS, one may achieve considerable gains in performance using the same marker set. The developed markers provide a cost-efficient tool for genotyping populations of tree of heaven and the approach presented here promises to be of high value for medium throughput genotyping applications in non-model forest tree species. We will use this method to widen the perspectives for further population genetic investigations of the tree of heaven.

Characterization of pelvic and cervical microbiotas from patients with pelvic inflammatory disease.

The aims of this work were: (i) to profile and compare pelvic and cervical microbiota from patients with pelvic inflammatory disease (PID); (ii) to test the ascending infection hypothesis that the microbes in the vagina or cervix spread to the upper genital tract and cause PID. Thirty-eight PID patients and 19 control patients were enrolled in this study and received salpingectomy or salpingo-oophorectomy. Both pelvic and cervical samples were collected during surgery, which were subject to culture diagnosis and next-generation sequencing (NGS)-based 16S rRNA profiling. For the PID group, the NGS-based method revealed that half of the pelvic samples were dominated by a single organism while the other half exhibited a polymicrobial infection. The pelvic and cervical microbiota were similar in terms of both taxonomic richness and evenness. Pelvic microbiota was dominated by Acinetobacter, Escherichia, Sneathia and Streptococcus whereas cervical microbiota was dominated by Lactobacillus and Gardnerella. Only six pelvic samples were positive by culture diagnosis, three of which matched the NGS-based results. The PID group was further divided into 23 hydrosalpinx and 15 pyosalpinx samples according to the pelvic mass collected during surgery, the former showing a richer bacterial composition than the latter. The NGS-based method provides a more informative profile of pelvic microbiota than the culture method. The pelvic and cervical microbiota from the same patient were inconsistent with each other, and thus therapeutic decisions based on cervical microbiota may lead to antimicrobial misuse.

Diagnostic Single Gene Analyses Beyond Sanger

SummaryMolecular testing of congenital coagulation and platelet disorders offers confirmation of clinical diagnoses, supports genetic counselling, and enables predictive and prenatal diagnosis. In some cases, genotype-phenotype correlations are important for predicting the clinical course of the disease and adaptation of individualized therapy. Until recently, genotyping has been mainly performed by Sanger sequencing. While next generation sequencing (NGS) enables the parallel analysis of multiple genes, the cost-value ratio of custom-made panels can be unfavorable for analyses of specific small genes. The aim of this study was to transfer genotyping of small genes involved in congenital coagulation and platelet disorders from Sanger sequencing to an NGS-based method. A LR-PCR approach for target enrichment of the entire genomic regions of the genes F7, F10, F11, F12, GATA1, MYH9, TUBB1 and WAS was combined with high-throughput sequencing on a MiSeq platform. NGS detected all variants that had previously been identified by Sanger sequencing. Our results demonstrate that this approach is an accurate and flexible tool for molecular genetic diagnostics of single small genes.

Introduction of a novel, universal NGS-based research Method for preimplantation genetic diagnosis and screening

Introduction: Over the past few years, several new technologies have been developed for the high resolution molecular cytogenetic analysis of embryos prior to implantation. Although technologies such as STR-PCR, SNP arrays and aCGH have been utilized for the detection of mutations associated with Single Gene Disorder (SGD) and/or translocations for Preimplantation Genetic Diagnosis (PGD), significant challenges persist. To overcome these challenges, we have developed a novel NGS-based method coupled with specific algorithms that utilize parental haplotype information to detect SGD-associated mutations and translocations. Our approach can be successfully applied both to single blastomeres and trophectoderm cells, and does not require gene-specific spike-ins, thereby making it highly versatile assay. Furthermore, our approach can concurrently detect aneuploidies, thus combining PGD and Preimplantation Genetic Screening (PGS) in one method.

Detection of copy number variations by pair analysis using next-generation sequencing data in inherited kidney diseases.

Comprehensive genetic approaches for diagnosing inherited kidney diseases using next-generation sequencing (NGS) have recently been established. However, even with these approaches, we are still failing to detect gene defects in some patients who appear to suffer from genetic diseases. One of the reasons for this is the difficulty of detecting copy number variations (CNVs) using our current approaches. For such cases, we can apply methods of array-based comparative genomic hybridization (aCGH) or multiplex ligation and probe amplification (MLPA); however, these are expensive and laborious and also often fail to identify CNVs. Here, we report seven cases with CNVs in various inherited kidney diseases screened by NGS pair analysis. Targeted sequencing analysis for causative genes was conducted for cases with suspected inherited kidney diseases, for some of which a definitive genetic diagnosis had not been achieved. We conducted pair analysis using NGS data for those cases. When CNVs were detected by pair analysis, they were confirmed by aCGH and/or MLPA. In seven cases, CNVs in various causative genes of inherited kidney diseases were detected by pair analysis. With aCGH and/or MLPA, pathogenic CNV variants were confirmed: COL4A5 or HNF1B in two cases each, and EYA1, CLCNKB, or PAX2 in one each. We presented seven cases with CNVs in various genes that were screened by pair analysis. The NGS-based CNV detection method is useful for comprehensive screening of CNVs, and our results revealed that, for a certain proportion of cases, CNV analysis is necessary for accurate genetic diagnosis.

Multiple identification of most important waterborne protozoa in surface water used for irrigation purposes by 18S rRNA amplicon-based metagenomics.

Understanding waterborne protozoan parasites (WPPs) diversity has important implications in public health. In this study, we evaluated a NGS-based method as a detection approach to identify simultaneously most important WPPs using 18S rRNA high-throughput sequencing. A set of primers to target the V4 18S rRNA region of WPPs such as Cryptosporidium spp., Giardia sp., Blastocystis sp., Entamoeba spp, Toxoplasma sp. and free-living amoebae (FLA) was designed. In order to optimize PCR conditions before sequencing, both a mock community with a defined composition of representative WPPs and a real water sample inoculated with specific WPPs DNA were prepared. Using the method proposed in this study, we have detected the presence of Giardia intestinalis, Acanthamoeba castellanii, Toxoplasma gondii, Entamoeba histolytica and Blastocystis sp. at species level in real irrigation water samples. Our results showed that untreated surface irrigation water in open fields can provide an important source of WPPs. Therefore, the methodology proposed in this study can establish a basis for an accurate and effective diagnostic of WPPs to provide a better understanding of the risk associated to irrigation water.

Strain-level Determination of the Contribution of Gut Microbiota to the Development of Bacteremia in Patients Undergoing Stem Cell Transplantation

BackgroundInfection is a major preventable cause of transplant-related morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HCT). Bacteremia is the most common infectious complication in HCT, often occurring during periods of mucositis when the risk for microbial translocation from the intestine is increased. Prior research in HCT patients using 16S rRNA sequencing demonstrated that gut microbiota dominance by either Enterococcus spp. or Proteobacteria was associated with the development of bacteremia with Enterococcus spp. and Gram-negative organisms, respectively. No studies to date, however, have compared bacteremia isolates and gut microbiota samples at a strain-specific level using next-generation shotgun metagenomic sequencing (NGS).MethodsIn order to assess the degree of genetic similarity between bacteremia isolates and the gut microbiota, we identified patients who had undergone HCT at Stanford and developed a bacteremia between October 2015 and September 2016 for whom we had both saved blood culture isolates and stool samples within 30 days preceding bacteremia. We identified 15 patients from whom we had 17 bacteremia isolates, and performed NGS (Illumina HiSeq 4000) on stool and isolate DNA. We generated draft assemblies of isolate genomes using the SPAdes assembler, and aligned stool metagenomic reads to the draft isolate genomes using Bowtie2, filtering reads for perfect end-to-end alignment.ResultsEnteric gram-negative bacteremia isolates were identical to those in the gut microbiota, as has been demonstrated in prior studies using older strain-typing Methods. Surprisingly, we also identified gram-positive organisms that were identical in both the blood and stool prior to bacteremia, which challenges existing dogma regarding sources of gram-positive bacteremia-causing organisms.ConclusionUsing a highly sensitive and accurate NGS-based strain typing method, we provide evidence of translocation of organisms from the gut microbiota and subsequent bacteremia. The gut was confirmed as a source for both classic enteric gram-negative and classically non-enteric Gram-positive bacteremia in HCT patients. These findings may have implications for the origins of bacteremia in HCT patients previously classified as CLABSIs.Disclosures A. Bhatt, Janssen Human Microbiome Institute / Johnson and Johnson: Consultant, Consulting fee