Primer Extension Sequencing Research Articles

Combining E-ice-COLD-PCR and Pyrosequencing with Di-Base Addition (PDBA) Enables Sensitive Detection of Low-Abundance Mutations.

Detecting low-abundance mutations is of particular interest in the fields of biology and medical science. However, most currently available molecular assays have limited sensitivity for the detection of low-abundance mutations. Here, we established a platform for detecting low-level DNA mutations with high sensitivity and accuracy by combining enhanced-ice-COLD-PCR (E-ice-COLD-PCR) and pyrosequencing with di-base addition (PDBA). The PDBA assay was performed by selectively adding one di-base (AG, CT, AC, GT, AT, or GC) instead of one base (A, T, C, or G) into the reaction at a time during sequencing primer extension and thus enabling to increase the sequencing intensity. A specific E-ice-COLD-PCR/PDBA assay was developed for the detection of the most frequent BRAF V600E mutation to verify the feasibility of our method. E-ice-COLD-PCR/PDBA assay permitted the reliable detection of down to 0.007% of mutant alleles in a wild-type background. Furthermore, it required only a small amount of starting material (20pg) to sensitively detect and identify low-abundance mutations, thus increasing the screening capabilities in limited DNA material. The E-ice-COLD-PCR/PDBA assay was applied in the current study to clinical formalin-fixed paraffin-embedded (FFPE) and plasma samples, and it enabled the detection of BRAF V600E mutations in samples that appeared as a wild type using PCR/conventional pyrosequencing (CP) and E-ice-COLD-PCR/CP. E-ice-COLD-PCR/PDBA assay is a rapid, cost-effective, and highly sensitive method that could improve the detection of low-abundance mutations in routine clinical use.

Transcript-specific determinants of pre-mRNA splicing revealed through in vivo kinetic analyses of the 1st and 2nd chemical steps

We generate high-precision measurements of the invivo rates of both chemical steps of pre-mRNA splicing across the genome-wide complement of substrates in yeast by coupling metabolic labeling, multiplexed primer-extension sequencing, and kinetic modeling. We demonstrate that the rates of intron removal vary widely, splice-site sequences are primary determinants of 1st step but have little apparent impact on 2nd step rates, and the 2nd step is generally faster than the 1st step. Ribosomal protein genes (RPGs) are spliced faster than non-RPGs at each step, and RPGs share evolutionarily conserved properties that may contribute to their faster splicing. A genetic variant defective in the 1st step of the pathway reveals a genome-wide defect in the 1st step but an unexpected, transcript-specific change in the 2nd step. Our work demonstrates that extended co-transcriptional association is an important determinant of splicing rate, a conclusion at odds with recent claims of ultra-fast splicing.

Unique Polymorphisms at BCL11A, HBS1L-MYB and HBB Loci Associated with HbF in Kuwaiti Patients with Sickle Cell Disease.

Patients with sickle cell disease (SCD) in Kuwait have elevated HbF levels ranging from ~10–44%; however, the modulating factors are unclear. We investigated the association of single nucleotide polymorphisms (SNPs) at BCL11A, HBS1L-MYB and HBB with HbF levels in 237 Kuwaiti SCD patients, divided into 3 subgroups according to their HbF levels. Illumina Ampliseq custom DNA panel was used for genotyping and confirmed by arrayed primer extension or Sanger sequencing. In the BCL11A locus, the CC genotype of rs7606173 [χ2 = 16.5] and (GG) of rs10195871 [χ2 = 15.0] were associated with Hb-F1 and HbF-2 subgroups, unlike rs1427404-T [χ2 = 17.3], which showed the highest association across the three subgroups. HBS1L-MYB locus revealed 2 previously-described SNPs (rs66650371 [χ2 = 9.5] and rs35795442 [χ2 = 9.2]) and 2 previously-unreported SNPs, (rs13220662 [χ2 = 6.2] and rs1406811 [χ2 = 6.7]) that were associated with the HbF-3 subgroup, making this the key locus elevating HbF to the highest levels. HBB cluster variants were associated with lower levels of HbF (β = −1.1). We report four previously-unpublished variants showing significant association with HbF. Each of the three quantitative trait loci affects HbF levels differently; unique SNPs, especially in HBS1L-MYB, elevate HbF to the highest levels.

The Diagnosis of HbS Genotypes and Identification of β-Thalassemia Mutations in Patients with Hbsβ-Thalassemia Using Next Generation Sequencing

The conventional diagnosis of sickle cell disease (SCD) depends on RBC morphology, Hb electrophoresis and/or high-performance liquid chromatography (HPLC). However, these methods are fraught with ambiguities especially in areas where HbSβ-thalassemia is common. The distinguishing laboratory feature between patients with HbSS and HbSb-thalassemia is the level of HbA2. Unfortunately, when HbA2 is estimated with HPLC in patients with Sβthalassemia, it tends to be spuriously high because it co-migrates with glycated HbS. HbSβ-thalassemia accounts for 30 - 40% of the SCD patients being followed in Kuwait. Patients with a presumptive diagnosis of HbSS and/or Sβ-thalassemia, by HPLC were referred to the Hemoglobin Research Laboratory of Department of Pediatrics, Kuwait University for confirmatory molecular diagnosis. The DNA samples collected from 1994 to 2018 were recently subjected to NGS. DNA was isolated from peripheral leukocytes using the phenol-chloroform method. The Illumina Ampliseq custom DNA panel was used to genotype the DNA samples. For theHBBlocus, all β-globin mutations and variants were confirmed by arrayed primer extension (APEX) or Sanger sequencing methods. In addition, genotyping of modifier SNPs inBCL11Aon chromosome 2 and theHBS1L-MYBintergenic region on chromosome 6 was carried out to identify their influence on HbF expression among our patients. In all, 126 SNPs were genotyped. We hereby, report the false positive and false negative rates for the diagnosis of Sb-thalassemia with HPLC compared to NGS. We also report the spectrum of β-thal mutations among our patients with Sβ-thalassemia. The DNA samples were from 232 patients aged from 1 to 59 with a mean of 12.7 ± 11.2 years. Analysis of the initial HPLC diagnosis showed that 27.7% were reported to have Sβ-thalassemia, while after NGS assessment, 29.4% were found to truly fit the diagnosis. Three individuals who were diagnosed as HbSS turned out to be HbAS and 1 each turned out to be HbAA and β-thalassemia trait respectively. Twelve patients that were originally thought to be Sβ-thalassemia turned out to be HbSS, i.e. a false positive rate of 5%, while 30 who were thought to be SS, turned out to be Sβ-thalassemia, i.e. a false negative rate of 13%. A total of 12 mutations were identified in 61 Sβ-thalassemia patients. Of these, the most common were the β0 IVS-1 del 25 and the IVS-II-1 (G/A) in 9 (14.8%) each. The most common β+ mutation was the IVS-I-110 (G/A) in 8 (13.1%), followed by the IVS-I-5 (G/C) in 7 (11.5%). While the Sβ0-thalassemia patients had no HbA on HPLC, the Sβ+ were associated with varying concentrations of HbA, ranging from 0 for those carrying the IVS-I-5 (G/C) mutation to a mean of 15.5 +/- 6.2% in the patients with IVS-I-110 (G/A) and 25.1% in the Sβ++patient with IVS-I-6 (C/T). Indeed, HPLC is far from deal in detecting HbSβ-thalassemia with significant rates of false positivity and negativity. NGS is very versatile; it can interrogate thousands of genes simultaneously, making it ideal for use in SCD. The HbS genotype, β-thalassemia mutation, haplotype and different modifier polymorphisms can be determined in one run. It is therefore very useful for personalized diagnosis that can document prognostic factors, making for purposeful counseling and follow-up from an early age. Disclosures No relevant conflicts of interest to declare.

Abstract 4296: Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells

Abstract Background: Although interfering with the actions of microtubule-associated proteins (MAP’s) is successful against ovarian epithelial carcinoma (OVCA), knowledge of the MAP’s in OVCA cells is lacking. We sought to identify cancer-specific MAP’s in OVCA cells.Methods: Four human OVCA cell lines, primary OVCA tissue, ascitic cells and metastases from 9 patients and normal ovarian tissue from six women were compared to control human and rat brain tissue, uterine myomas (benign tumors) and rat and human non-neural organs. Modified Western blotting using panels of anti-microtubule-associated protein monoclonal antibodies were used. An in-vitro brain and OVCA mixing and recovery experiment was carried out to assess possible degradation of MAP1a by OVCA. A paclitaxel-driven tubulin assembly assay was performed to determine whether the OVCA protein has microtubule-associated protein properties. The transcriptome of CSP-80 was sequenced using primer extension sequencing. Results: (1) In contradistinction to the 350 kDa MAP1a present in control brain extracts, only an 80 kDa molecule with MAP1a immune-reactivity was found in OVCA cell lines and tissues. No immune-reactivity was present in the negative controls. (2)Following the mixing experiments there was no increase of CSP-80; CSP-80 is not a degradation artifact of MAP1a. (3) The paclitaxel-driven tubulin assembly assay showed that CSP-80 bound to microtubules during tubulin polymerization. (4) No CSP-80 was identified in human or rat non-malignant tissues including normal human and rat brain or normal human ovary, placenta or uterine myomas, or normal rat liver, spleen or lung. (5) cDNA sequencing shows a 2603 bp fragment at a 5’ terminal of p80 to be homologous with the analogous segment of MAP1a. (6) Further testing showed that CSP-80 was present in other reproductive and non-reproductive carcinomas. Again, no 350 kDa MAP1a was present. Studies are underway to determine whether CSP-80 is a mutated protein or the product of post-transcriptional modification. Conclusion: CSP-80 is a novel cancer-related protein that appears to be limited to an 80 kDa sequence of the MAP1a gene, or a post-transcriptionally modified product. Since CSP-80 exhibits tubulin-binding activity, this may explain the efficacy of taxane derivatives. CSP-80 may disclose some aspects of carcinogenesis, be a valuable marker of cancer and therapeutic efficacy, and offer new avenues for cancer treatment. Citation Format: Ahmed Fadiel, Viswam S. Nair, Peter Aceto, Frederick Naftolin. Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells [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 4296.

Multiplexed primer extension sequencing: A targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates.

The study of pre-mRNA splicing has been greatly aided by the advent of RNA sequencing (RNA-seq), which enables the genome-wide detection of discrete splice isoforms. Quantification of these splice isoforms requires analysis of splicing informative sequencing reads, those that unambiguously map to a single splice isoform, including exon-intron spanning alignments corresponding to retained introns, as well as exon-exon junction spanning alignments corresponding to either canonically- or alternatively-spliced isoforms. Because most RNA-seq experiments are designed to produce sequencing alignments that uniformly cover the entirety of transcripts, only a comparatively small number of splicing informative alignments are generated for any given splice site, leading to a decreased ability to detect and/or robustly quantify many splice isoforms. To address this problem, we have recently described a method termed Multiplexed Primer Extension sequencing, or MPE-seq, which uses pools of reverse transcription primers to target sequencing to user selected loci. By targeting reverse transcription to pre-mRNA splice junctions, this approach enables a dramatic enrichment in the fraction of splicing informative alignments generated per splicing event, yielding an increase in both the precision with which splicing efficiency can be measured, and in the detection of splice isoforms including rare splicing intermediates. Here we provide a brief review of the shortcomings associated with RNA-seq that drove our development of MPE-seq, as well as a detailed protocol for implementation of MPE-seq.

Molecular Switch Controlling Expression of the Mannose-Specific Adhesin, Msa, in Lactobacillus plantarum.

Some lactic acid bacteria, especially Lactobacillus spp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probiotic Lactobacillus plantarum strains, WCSF1 and 299v, display highly different mannose-specific adhesion, with L. plantarum 299v being superior to L. plantarum WCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of the msa gene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a stronger msa transcript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of the msa gene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate the msa transcript. In addition, the ribosome binding site upstream of the msa gene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of the msa transcript in the variants than in the wild type, correlating with a strong-Msa phenotype.IMPORTANCE Probiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. In Lactobacillus plantarum, interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of the msa gene in L. plantarum strain WCFS1. Strains with increased msa expression could be valuable in the development and manufacture of improved probiotic products.

Induced Protein-RNA Interactions in the 5’ UTR Lead to Conformational Changes that Alter Translation

We study translational regulation by a 5’ UTR sequence encoding the binding site of an RNA-binding protein (RBP) in bacteria, using a reporter assay and selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-Seq). We tested constructs containing a single hairpin, based on the binding sites of the coat RBPs of bacteriophages GA, MS2, PP7, and Qβ, positioned in the 5’ UTR of a reporter gene. With specifically-bound RBP present, either weak repression or up-regulation is observed, depending on the binding site and its flanking sequence. SHAPE-Seq data for a representative construct exhibiting up-regulation, indicates a partially-folded hairpin and non-reactive upstream and downstream flanking region, which we attribute to intermediate structures that apparently block translation. RBP binding stabilizes the fully-folded hairpin state facilitating translation, suggesting that the up-regulating constructs function as protein-sensing regulatory elements. This finding is further supported by lengthening the binding-site stem, which in turn destabilizes the translationally-inactive state, and abolishes the up-regulating behavior. Finally, we find that the combination of two binding sites, positioned in the 5’ UTR and the ribosomal initiation region within the CDS of the same transcript, can yield a cooperative regulatory response. Together, we show that the interaction of an RBP with its RNA target facilitates structural changes in the RNA, which is reflected by a controllable range of binding affinities and dose response behaviors. Thus, demonstrating that RNA-RBP interactions can provide a platform for constructing gene regulatory networks that are based on translational, rather than transcriptional, regulation.

Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements.

In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3′ untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites.

The Sickle β-Thalassemia Phenotype.

Sβ-thalassemia (Sβ-thal) is common among Gulf Arab patients with sickle cell disease, but the phenotype of this group had not been well-documented. We have studied a group of Kuwaiti patients and compared the phenotype in the homozygotes (SS) and Sβ-thal patients. Complete blood count, hemoglobin quantitation, serum bilirubin, and lactate dehydrogenase were determined with standard techniques. The patients were screened for α-globin genotype. The Sβ-thal patients were also screened for the HBG2 Xmn-1 polymorphism. β-Thal mutations were determined by arrayed primer extension or direct sequencing. There were 70 SS and 32 Sβ-thal patients with mean ages of 14.8±5.9 and 14.2±5.9 years, respectively. The Sβ-thal patients had more frequent, severe pain episodes per year compared with the SS, while the patterns among Sβ-thal and Sβ-thal patients were not significantly different. There were no differences in the frequencies of acute chest syndrome, gallstones, and blood transfusion in the SS and Sβ-thal patients. However, none of the Sβ-thal patients had been transfused. Among the Sβ-thal patients, 25 had β-thal and 7 had β-thal mutations, the most common being cd39 (C→T) and IVS-I-110 (G→A), respectively. Sβ-thal shows a severe phenotype in Kuwait, even among those with Sβ-thal, in whom the IVS-I-110 (G→A) mutation is predominant.

Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin–streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin–SAv roadblocks. We then show that randomly distributed biotin–SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin–SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq.

Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation.

The molecular properties of the phosphodiester backbone that made it the evolutionary choice for the enzymatic replication of genetic information are not well understood. To address this, and to develop new chemical ligation strategies for assembly of biocompatible modified DNA, we have synthesized oligonucleotides containing several structurally and electronically varied artificial linkages. This has yielded a new highly promising ligation method based on amide backbone formation that is chemically orthogonal to CuAAC "click" ligation. A study of kinetics and fidelity of replication through these artificial linkages by primer extension, PCR, and deep sequencing reveals that a subtle interplay between backbone flexibility, steric factors, and ability to hydrogen bond to the polymerase modulates rapid and accurate information decoding. Even minor phosphorothioate modifications can impair the copying process, yet some radical triazole and amide DNA backbones perform surprisingly well, indicating that the phosphate group is not essential. These findings have implications in the field of synthetic biology.

Cotranscriptional folding of a riboswitch at nucleotide resolution.

RNAs can begin to fold immediately after emerging from RNA polymerase during transcription. Interactions between nascent RNAs and ligands during cotranscriptional folding can direct the formation of alternative RNA structures, a feature exploited by non-coding RNAs called riboswitches to make gene regulatory decisions. Despite their importance, cotranscriptional folding pathways have yet to be uncovered with sufficient resolution to reveal how cotranscriptional folding governs RNA structure and function. To access cotranscriptional folding at nucleotide resolution, we extend selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to measure structural information of nascent RNAs during transcription. With cotranscriptional SHAPE-Seq, we determine how the B. cereus crcB fluoride riboswitch cotranscriptional folding pathway undergoes a ligand-dependent bifurcation that delays or promotes terminator formation via a series of coordinated structural transitions. Our results directly link cotranscriptional RNA folding to a genetic decision and establish a framework for cotranscriptional analysis of RNA structure at nucleotide resolution.

Understanding the Functions of Long Non-Coding RNAs through Their Higher-Order Structures.

Although thousands of long non-coding RNAs (lncRNAs) have been discovered in eukaryotes, very few molecular mechanisms have been characterized due to an insufficient understanding of lncRNA structure. Therefore, investigations of lncRNA structure and subsequent elucidation of the regulatory mechanisms are urgently needed. However, since lncRNA are high molecular weight molecules, which makes their crystallization difficult, obtaining information about their structure is extremely challenging, and the structures of only several lncRNAs have been determined so far. Here, we review the structure–function relationships of the widely studied lncRNAs found in the animal and plant kingdoms, focusing on the principles and applications of both in vitro and in vivo technologies for the study of RNA structures, including dimethyl sulfate-sequencing (DMS-seq), selective 2′-hydroxyl acylation analyzed by primer extension-sequencing (SHAPE-seq), parallel analysis of RNA structure (PARS), and fragmentation sequencing (FragSeq). The aim of this review is to provide a better understanding of lncRNA biological functions by studying them at the structural level.

Characterizing RNA structures in vitro and in vivo with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

RNA molecules adopt a wide variety of structures that perform many cellular functions, including, among others, catalysis, small molecule sensing, and cellular defense. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally, we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

Abstract 01: Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS)

Abstract Background: AML is a clinically and genetically heterogeneous clonal disorder. Approximately 50% are characterized by recurrent clonal chromosome aberrations which have contributed to the classification of disease and are recognized as important prognostic factors. AML patients who lack these recurrent structural abnormalities have been grouped as “intermediate cytogenetic risk” and are being further subcategorized by the sequence alterations that are being identified. A more complete understanding of the genetic changes that are relevant to the pathogenesis of AML will improve the classification of risk and ultimately better selection of therapy. Our hypothesis is that mutational profiling and analysis of patient outcomes will help better define the risk subgroups of patients and predict prognosis in patients with AML. Methods: Archived DNA from 37 patients with various AML diagnoses were obtained with IRB approval (IRB#201502763). A panel of 30 commonly mutated genes in AML were designed using the Ion Ampliseq Designer (Ion Torrent) and targeted for “hotspot” or complete coding sequence (CDS). Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) technology. Library clonal amplification was performed by emulsion PCR using Ion PGM™ Template OT2 Reagents Kit200. Simultaneous sequencing of clonally amplified DNA targets was performed by Ion-Torrent Personalized Genome Machine (IT-PGM) using 318v2 chip. Genomic variants were identified by variant caller3.4 and further filtered and analysed through a custom-made clinically validated pipeline. Novel mutations were confirmed by either single nucleotide primer extension or Sanger sequencing. Results: The assay successfully identified previously identified point mutations in FLT-3 and CEBPA genes. Additional variants were identified in 81% (30/37) of specimens; no mutations were found in 19% (7/37) specimens. The genes most frequently mutated were DNMT3A (23%), TET2 (17%), IDH2 (14%) and TP53 (14%). U2AF1 (11%) and RUNX1 (8%) were exclusively mutated in specimens with non-recurrent cytogenetic abnormalities. The identified variants will be used to risk stratify patients based on clinical outcome. Conclusion: This targeted multi-gene panel successfully identified several variants along with previously known mutations in archived specimens. Citation Format: Anup Tilak, Ramakrishna Sompallae, Aaron Stence, Natalya Guseva, Aaron Bossler. Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS). [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 01.

SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing.

RNA structure is a primary determinant of its function, and methods that merge chemical probing with next generation sequencing have created breakthroughs in the throughput and scale of RNA structure characterization. However, little work has been done to examine the effects of library preparation and sequencing on the measured chemical probe reactivities that encode RNA structural information. Here, we present the first analysis and optimization of these effects for selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). We first optimize SHAPE-Seq, and show that it provides highly reproducible reactivity data over a wide range of RNA structural contexts with no apparent biases. As part of this optimization, we present SHAPE-Seq v2.0, a ‘universal’ method that can obtain reactivity information for every nucleotide of an RNA without having to use or introduce a specific reverse transcriptase priming site within the RNA. We show that SHAPE-Seq v2.0 is highly reproducible, with reactivity data that can be used as constraints in RNA folding algorithms to predict structures on par with those generated using data from other SHAPE methods. We anticipate SHAPE-Seq v2.0 to be broadly applicable to understanding the RNA sequence–structure relationship at the heart of some of life's most fundamental processes.

A Practical Comparison of Ligation-Independent Cloning Techniques

The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.

SeqFold: Genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data

We present an integrative approach, SeqFold, that combines high-throughput RNA structure profiling data with computational prediction for genome-scale reconstruction of RNA secondary structures. SeqFold transforms experimental RNA structure information into a structure preference profile (SPP) and uses it to select stable RNA structure candidates representing the structure ensemble. Under a high-dimensional classification framework, SeqFold efficiently matches a given SPP to the most likely cluster of structures sampled from the Boltzmann-weighted ensemble. SeqFold is able to incorporate diverse types of RNA structure profiling data, including parallel analysis of RNA structure (PARS), selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), fragmentation sequencing (FragSeq) data generated by deep sequencing, and conventional SHAPE data. Using the known structures of a wide range of mRNAs and noncoding RNAs as benchmarks, we demonstrate that SeqFold outperforms or matches existing approaches in accuracy and is more robust to noise in experimental data. Application of SeqFold to reconstruct the secondary structures of the yeast transcriptome reveals the diverse impact of RNA secondary structure on gene regulation, including translation efficiency, transcription initiation, and protein-RNA interactions. SeqFold can be easily adapted to incorporate any new types of high-throughput RNA structure profiling data and is widely applicable to analyze RNA structures in any transcriptome.

Mitochondrial Haplogroups and Control Region Polymorphisms in Age-Related Macular Degeneration: A Case-Control Study

BackgroundOnset and development of the multifactorial disease age-related macular degeneration (AMD) are highly interrelated with mitochondrial functions such as energy production and free radical turnover. Mitochondrial dysfunction and overproduction of reactive oxygen species may contribute to destruction of the retinal pigment epithelium, retinal atrophy and choroidal neovascularization, leading to AMD. Consequently, polymorphisms of the mitochondrial genome (mtDNA) are postulated to be susceptibility factors for this disease. Previous studies from Australia and the United States detected associations of mitochondrial haplogroups with AMD. The aim of the present study was to test these associations in Middle European Caucasians.Methodology/Principal FindingsMitochondrial haplogroups (combinations of mtDNA polymorphisms) and mitochondrial CR polymorphisms were analyzed in 200 patients with wet AMD (choroidal neovascularization, CNV), in 66 patients with dry AMD, and in 385 controls from Austria by means of multiplex primer extension analysis and sequencing, respectively. In patients with CNV, haplogroup H was found to be significantly less frequent compared to controls, and haplogroup J showed a trend toward a higher frequency compared to controls. Five CR polymorphisms were found to differ significantly in the two study populations compared to controls, and all, except one (T152C), are linked to those haplogroups.Conclusions/SignificanceIt can be concluded that haplogroup J is a risk factor for AMD, whereas haplogroup H seems to be protective for AMD.