Sequencing Chromatograms Research Articles

Novel splicing variants of recepteur d'origine nantais (RON) tyrosine kinase involving exons 15–19 in lung cancer

BackgroundAltered expressions of receptor tyrosine kinases drive the growth and metastasis of several cancers. RON is a single pass transmembrane receptor tyrosine kinase (RTK) shown to be aberrantly expressed in various cancer types. However, target validation and successful therapeutic targeting of RON in cancers is hampered by the co-existence of unknown number/types of isoforms, which are structurally similar but functionally diverse. ObjectiveThe objective of this study was to identify differential splicing in the C-terminal region of RON transcripts to better understand RON signaling in cancers. mRNA transcript sequence between exons 14 and 20 of RON was PCR amplified and sequenced using cDNA from 10 SCLC and 13 NSCLC cell lines. Specific exon deletions were identified by aligning sequencing chromatograms with reference RON cDNA sequence. ResultsWe identified the presence of four unique transcript sequence variants of RON formed through skipping of exons 15–19, 16–19, 16–17 and 16. The transcript variants, except the one lacking exons 15–19, were found in more than one cell line. Several cell lines contained two to four of these uniquely spliced transcript variants. dbEST (Expressed Sequence Tags database) or other DNA sequence databases did not contain RON cDNA sequences corresponding to any of the above exon deletions indicating that all these transcript sequence alterations are novel. ConclusionsResults of our study indicate common occurrence of different types of alternatively spliced transcripts of RON in lung cancer with potential to be translated into proteins lacking active kinase domain. Our findings suggest that tumors produce several dominant negative isoforms which probably inhibit ligand dependent RON signaling, and hence, raise important questions regarding the appropriateness of blocking wild type RON signaling for therapy. Further, presence of transcript variants and their isoform products may interfere with quantitative and functional analysis during target validation.

Identification of a Novel SLC40A1 Arg88Ile Mutation in a Patient with Familial Iron Overload Treated By Phlebotomy

INTRODUCTIONThe cellular iron exporter ferroportin, encoded by the SLC40A1 gene, plays a key role in systemic iron regulation by mediating the absorption of dietary iron from duodenal enterocytes and the release of macrophage iron stores into the plasma. SLC40A1 mutations result in a clinically heterogeneous iron overload disorder exhibiting autosomal dominant transmission. Mutations that impair iron export function result in a classical ferroportin disease phenotype characterized by hyperferritinemia, normal transferrin saturation, and macrophage iron loading, while mutations that impair the regulation of ferroportin by hepcidin result in a non-classical form of disease exhibiting high transferrin saturation and hepatocellular iron loading. Here we report the clinical phenotype of a male patient with a personal and family history of iron overload who was found to harbor a novel SLC40A1 mutation. CLINICAL HISTORYA 39-year-old male of Italian descent came to clinical attention after laboratory evidence of iron overload was detected at the time of a routine physical exam. Serum ferritin was markedly elevated at 5018 ng/mL, while transferrin iron saturation was within the normal range at 42%. A complete blood count revealed hemoglobin 16.1 g/dL, hematocrit 48.7%, and MCV 96.7 fL. Liver function tests revealed mild elevation of transaminases (AST 50 U/L, ALT 114 U/L). HBV and HCV serologies, as well as an anti-nuclear antibody screen, were negative. Rheumatoid factor, ceruloplasmin, and alpha-fetoprotein were within the normal range. Genetic testing for the HFE C282Y, H63D, and S65C variants was negative. Abdominal ultrasound revealed a somewhat course and echogenic liver. The patient's past medical and surgical histories were non-contributory. Family history was notable for a 63-year-old father with non-HFE hemochromatosis treated by phlebotomy, as well as a possible history of iron overload in the paternal grandmother. There was no known family history of hepatocellular carcinoma. The patient reported consuming ≤ 5 alcoholic beverages per week. Review of systems was negative, and no organomegaly was detected on physical exam. The patient began undergoing phlebotomy approximately every 2 weeks, which now has been well tolerated for almost two years. His most recent ferritin level was within the normal range (328 ng/mL). METHODSUsing genomic DNA extracted from peripheral blood as template, all coding regions and intron-exon boundaries of SLC40A1 were amplified by polymerase chain reaction and analyzed by bidirectional Sanger sequencing. Sequence chromatograms were analyzed using Sequencher software. This study was approved by the Yale University Human Investigation Committee (protocol #010412377). RESULTSA heterozygous single nucleotide substitution (c.263G>T, encoding p.Arg88Ile) was identified in exon 3 of the SLC40A1 gene (nomenclature per Ensembl reference transcript ENST00000261024). Ferroportin is a predicted multipass transmembrane protein, and Arg88 resides between two predicted transmembrane domains. Protein sequence alignments reveal that amino acid 88 is conserved as an arginine in ferroportin homologs in species as evolutionarily distant as Xenopus laevis and Danio rerio, suggesting that this residue is required for normal ferroportin function. The p.Arg88Ile variant has not been reported in the 1000 Genomes Project or the Exome Aggregation Consortium, demonstrating that it is not a polymorphism in the general population. The mutation predictor algorithms PolyPhen2, SIFT, and MutationTaster all strongly predicted this mutation to be damaging. DISCUSSIONThe ferroportin variant detected in this case (p.Arg88Ile) represents the third non-synonymous substitution at ferroportin residue 88 detected in patients with iron overload phenotypes. p.Arg88Gly was identified in a 38-year-old male who became anemic under a phlebotomy program (Cunat S., et al., Clin Chem 2007), while p.Arg88Thr was detected in multiple affected members of a single kindred in whom serial phlebotomy was well tolerated (Bach V, et al. Blood Cells Mol Dis 2006). Collectively, these findings suggest that the particular amino acid substitution at residue 88 may influence the degree of cellular iron sequestration. Future work will assess the effect of the Arg88Ile substitution on ferroportin function. DisclosuresNo relevant conflicts of interest to declare.

Corrigendum to "Congenital microcephaly and chorioretinopathy due to de novo heterozygous KIF11 mutations: Five novel mutations and review of the literature. Am J Med Genet Part A 2014 164A:2879-86".

In Tables I and II in the article, there are a few errors in the genomic coordinates and mutation nomenclature. Revised Table II lists the mutations with the correct nomenclature. Supplementary Figure I shows representative Sanger sequencing chromatograms of these mutations. The authors regret these errors. These mutations have been submitted to the LOVD KIF11 database (http://databases.lovd.nl/shared/variants/KIF11). The individual IDs of these mutations are 47821, 47847, 47849, 47850 and 47851. Additional supporting information may be found in the online version of this article at the publisher's web-site. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A multicenter comparison study on the detection of BCR-ABL tyrosine kinase domain point mutation

目的调查不同医院BCR-ABL酪氨酸激酶区点突变检测的准确性和一致性。方法由6家医院各制备10份来自于酪氨酸激酶抑制剂耐药的BCR-ABL（+）患者骨髓或外周血样本的cDNA，每份分装成6管派发，每家医院按照自身的操作程序检测共60份比对样本的BCR-ABL激酶区点突变，由北京大学人民医院结合测序图谱进行结果比对分析。结果37份（61.7%）比对样本的碱基及相应氨基酸突变类型6家医院报告均一致。53份样本可以确定突变类型，共涵盖23种；1份样本没有突变；6份样本由于室间图谱差异较大无法明确结果。突变比例低造成12份样本结果不一致，二者明显相关（P=0.008）；测序图谱特殊及扩增区域未覆盖各造成1份样本结果不一致；3份样本发生扩增失败；7份样本发生检测或报告错误。80.6％的结果明确的样本室间报告突变比例差值在20％以内。标本内参弱与检测失败和室间测序图谱差异大均相关。结论通过样本比对能够发现临床常规检测存在的问题，促进检测水平的提高。突变比例低是导致室间突变报告不一致的主要因素。

First Identification of Compound Heterozygous FKRP Mutations in a Korean Patient with Limb-Girdle Muscular Dystrophy.

Dystroglycanopathies are a genetically and clinically heterogeneous group of muscular dystrophies that are characterized by reduced glycosylation of α-dystroglycan. Among 18 causative genes, the fukutin-related protein (FKRP; MIM 606596) gene is the most common cause in Caucasians.1 However, FKRP mutations have rarely been found in the Asian population,2 and have not been reported previously in Korea. Herein we report novel compound FKRP mutations in a patient with myopathy, which represents the first reported case in Korea. A 43-year-old female (II-4) (Fig. 1A) was the fourth child of nonconsanguineous healthy Korean parents who achieved normal motor milestones after birth. She first noticed difficulty in climbing stairs at age 7 years, but muscle weakness did not progress. At the age of 23 years the patient perceived the progression of muscle weakness and was admitted to our institute. She displayed proximal weakness, especially in the pelvic girdle muscles. Mild winged scapulae and lordosis were also observed. No sensory deficits, joint contracture, tongue hypertrophy, or mental retardation were apparent. Her serum creatine kinase level was markedly elevated, at 2,439 IU/L (reference value: <185 IU/L). An electrophysiological study revealed a generalized myogenic process. No cardiac abnormality was observed. At the last examination, when she was 43 years old, she could walk with assistance. Fig. 1 Pedigree, sequencing chromatograms, and conservation profile of the patient. A: Pedigree of a Korean patient with compound heterozygous FKRP mutations. The filled symbol indicates the affected member; open symbols indicate unaffected members. B: Sequencing ... The genetic cause of this patient's myopathy was evaluated by performing targeted sequencing of 69 myopathy-causative genes including FKRP (Supplementary Information, Supplementary Table 1, 2, and 3 in the online-only Data Supplement). DNA fragments in the target regions were enriched by solution-based hybridization capture, followed by sequencing with an Illumina Hiseq2000 platform. The sequencing data were analyzed using a standard pipeline. Variants were then filtered further based on the patient's phenotype. Compound heterozygous FKRP mutations of c.857G>C and c.1170_1171delGC were identified (Fig. 1B). The c.1170_1171delGC mutation has been reported previously in a Japanese patient with congenital muscular dystrophy.2 However, the c.857G>C mutation was novel, and was not detected in 352 healthy controls, dbSNP138, or the 1000 Genomes Database (September 2014 release). In silico analysis using SIFT and PolyPhen2 predicted this mutation to be disease-causing. Genomic evolutionary rate profiling (GERP) indicated that the affected nucleotide is highly conserved (score=4.16), as became apparent upon inspection of the amino acid sequence in the altered region of the protein (Fig. 1C). Thus, the compound heterozygous FKRP mutations of c.1170_1171delGC and c.857G>C were determined to be the underlying cause of the myopathy in this patient. FKRP mutations result in various clinical presentations from severe brain abnormality to mild limb-girdle muscular dystrophy.1 This variability makes it difficult to diagnose FKRP-related muscular dystrophy based on clinical findings alone. Therefore, a step-by-step, classical approach with assessment of muscle pathology, muscle immunoanalysis, and mutational analysis is needed for the diagnosis of FKRP-related muscular dystrophy. The findings of a recent study in Taiwan that employed this approach suggested that FKRP-related muscular dystrophy is underdiagnosed, and not rare in the Asian population.3 However, this classical approach often fails to identify the causative gene due to pathological variability and the limited power of muscle immunoanalysis. Advances in next-generation sequencing, including targeted sequencing, have enabled the rapid and cost-effective analysis of many causative genes in uncategorized muscular dystrophy.4 In the present case, compound heterozygous FKRP mutations were identified using such next-generation sequencing. In conclusion, novel compound heterozygous FKRP mutations were identified in a patient with limb-girdle muscular dystrophy; this is the first reported case in Korea.

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses.

BackgroundMembers of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported.MethodsThe 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains.ResultsHeterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.ConclusionSequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the inter-species similarities were relatively low, ranging from 68.7–97.9%. The housekeeping genes rpoB and gyrB1 were demonstrated to be alternative classification markers to the species level based on intra- and inter-species comparisons, whereas based on phylogenetic tree rpoB proved to be reliable phylogenetic marker for the genus Prevotella.

An RNA editing fingerprint of cancer stem cell reprogramming.

BackgroundDeregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populations can be technically challenging, costly and requires PCR validation. The objectives of this study were to validate RNA editing of a subset of cancer stem cell-associated transcripts, and to develop a quantitative RNA editing fingerprint assay for rapid detection of aberrant RNA editing in human malignancies.MethodsTo facilitate quantification of cancer stem cell-associated RNA editing in exons and intronic or 3'UTR primate-specific Alu sequences using a sensitive, cost-effective method, we established an in vitro RNA editing model and developed a sensitive RNA editing fingerprint assay that employs a site-specific quantitative PCR (RESSq-PCR) strategy. This assay was validated in a stably-transduced human leukemia cell line, lentiviral-ADAR1 transduced primary hematopoietic stem and progenitor cells, and in primary human chronic myeloid leukemia stem cells.ResultsIn lentiviral ADAR1-expressing cells, increased RNA editing of MDM2, APOBEC3D, GLI1 and AZIN1 transcripts was detected by RESSq-PCR with improved sensitivity over sequencing chromatogram analysis. This method accurately detected cancer stem cell-associated RNA editing in primary chronic myeloid leukemia samples, establishing a cancer stem cell-specific RNA editing fingerprint of leukemic transformation that will support clinical development of novel diagnostic tools to predict and prevent cancer progression.ConclusionsRNA editing quantification enables rapid detection of malignant progenitors signifying cancer progression and therapeutic resistance, and will aid future RNA editing inhibitor development efforts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0370-3) contains supplementary material, which is available to authorized users.

Design and validation of a clinical DNA sequencing assay for drug-resistance genes in hepatitis B virus

Objective Under the ISO15189 and America Association of Pathologists (CAP) laboratory accreditation system, to establish the performance verification standards for detecting hepatitis B virus resistance gene by sanger sequencing. Methods 25 cases of HBV drug resistance outpatients and inpatients were collected from August 2012 to December in Hepatitis Clinic of Huashan Hospital Affiliated to Fudan University. Analytical performance parameters including analytical sensitivity, precision, accuracy, analytical specificity, reference range/reportable range, etc were evaluated. Sequencing quality evaluation parameters included fluorescence signal intensity overall sequencing chromatogram, signal to noise ratio, trace scores and QV value. Results 10%-20% mutation could be detected under wild-type background. The methool had good precision and accuracy.No obvious interference and cross contamination were observed. Conclusions Performance validation of the sequencing should combine with the practical application. Especially in view of the different detection subjects, and appropriately adjusted to meet the clinical needs. Detection of hepatitis B virus resistance gene by the in the test method in this study can be used in clinical detection.（Chin J Lab Med，2014,37：776-779） Key words: Hepatitis B virus; Drug resistance, viral; DNA, viral; Viral load; Sequence analysis, DNA; Polymerase chain reaction

A novel nonsense mutation in the sedlin gene (SEDL) causes severe spondyloepiphyseal dysplasia tarda in a five-generation Chinese pedigree

Spondyloepiphyseal dysplasia tarda (SEDT) is an X-linked recessive osteochondrodysplasia characterized by disproportionately short stature and degenerative joint disease. The objective of this study was to describe a novel nonsense mutation in the sedlin gene (SEDL) causing severe SEDT in a large Chinese pedigree. The clinical features of all affected individuals and female carriers were presented. Four affected males of the family were diagnosed with SEDT according to their clinical and radiological features. Direct DNA sequencing of SEDL was performed. Reverse-transcription polymerase chain reaction (RT-PCR) experiments of total RNA from blood lymphocytes were performed to confirm the defect in SEDL. DNA sequencing revealed that all of the affected males carried a nonsense mutation (c.61G>T) in SEDL that has not been previously reported. The c.61G>T mutation resulted in a premature translation termination codon (GAG>TAG) at amino acid position 21 (p.E21*), and was predicted to initiate the degradation of mutant transcripts through the nonsense-mediated mRNA decay pathway. Two female carriers showed typical sequencing chromatograms of a heterozygote. Following genetic counseling, individual IV7 gave birth to a healthy baby. Therefore, identification of the novel nonsense mutation (c.61G>T) in the SEDT family enables carrier detection, genetic counseling, and prenatal diagnosis. The detailed genotype/phenotype descriptions contribute to the SEDL mutation spectrum. The continued identification of mutations in SEDT patients will greatly aid further elucidation of the role of the sedlin protein in normal bone growth.

Inferring short-range linkage information from sequencing chromatograms.

Direct Sanger sequencing of viral genome populations yields multiple ambiguous sequence positions. It is not straightforward to derive linkage information from sequencing chromatograms, which in turn hampers the correct interpretation of the sequence data. We present a method for determining the variants existing in a viral quasispecies in the case of two nearby ambiguous sequence positions by exploiting the effect of sequence context-dependent incorporation of dideoxynucleotides. The computational model was trained on data from sequencing chromatograms of clonal variants and was evaluated on two test sets of in vitro mixtures. The approach achieved high accuracies in identifying the mixture components of 97.4% on a test set in which the positions to be analyzed are only one base apart from each other, and of 84.5% on a test set in which the ambiguous positions are separated by three bases. In silico experiments suggest two major limitations of our approach in terms of accuracy. First, due to a basic limitation of Sanger sequencing, it is not possible to reliably detect minor variants with a relative frequency of no more than 10%. Second, the model cannot distinguish between mixtures of two or four clonal variants, if one of two sets of linear constraints is fulfilled. Furthermore, the approach requires repetitive sequencing of all variants that might be present in the mixture to be analyzed. Nevertheless, the effectiveness of our method on the two in vitro test sets shows that short-range linkage information of two ambiguous sequence positions can be inferred from Sanger sequencing chromatograms without any further assumptions on the mixture composition. Additionally, our model provides new insights into the established and widely used Sanger sequencing technology. The source code of our method is made available at http://bioinf.mpi-inf.mpg.de/publications/beggel/linkageinformation.zip.

Minor Subclones Harboring Small Insertions and Deletions Probably Due To Aberrant Splicing Can Frequently Be Detected By Deep Sequencing of The BCR-ABL Kinase Domain

Background and Aimalthough BCR-ABL kinase domain (KD) mutations can frequently be identified in patients who develop resistance to tyrosine kinase inhibitors (TKIs) in Philadelphia-positive (Ph+) leukemias, other mechanisms may play a role. Small insertions and deletions within the BCR-ABL KD have occasionally been reported in chronic myeloid leukemia (CML) patients who failed TKI therapy and have been hypothesized to have a causative role in drug resistance. Some were in-frame insertions and deletions, others were predicted to result in truncated BCR-ABL proteins. However, the detection of these sequence variations is hampered by the fact that they are almost always confined to subclones co-exisiting with full length BCR-ABL. This has most likely resulted in an underestimation of their frequency and complexity, since i) they can be confused with background noise/reduced quality readings in direct sequencing chromatograms and ii) cloning would be needed to better resolve overlapping sequences in these samples. The recent development of Deep Sequencing (DS) technologies has opened the way to a more accurate characterization of molecular aberrations. DS enables greater sensitivity, quantitation of sequence variant abundance and clonal analysis of a given DNA region. We thus took advantage of DS to better characterize the spectrum of insertions and deletions in CML and Ph+ acute lymphoblastic leukemia (ALL) patients with response or resistance to TKI therapy. Methodsa total of 110 samples of 41 CML and 16 Ph+ ALL patients who received one or multiple lines of TKI therapy were analyzed. DS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study. Runs were designed to achieve high sequencing depth; this allowed to reliably identify and characterize deletions or insertions with a lower detection limit of 0.1%. In order to reconstruct the dynamics of evolution of these sequence variations in relation to the TKI administered and to the level of response achieved, we evaluated their presence in serial follow-up samples collected during TKI therapy in 15 patients. ResultsDS revealed a 35-base pair (bp) insertion in 35/41 (85%) CML and 14/16 (87%) Ph+ ALL patients. This sequence variation, already reported in the literature as ‘35INS', consists in the retention of 35 nucleotides (nt) from intron 8 at the exon 8 to exon 9 border. It leads to a truncated BCR-ABL variant having 10 a.a. encoded by intron 8 sequences but lacking 653 C-terminal a.a., including 22 a.a. of the KD, along with the entire C-terminal region. 35INS was detected with variable abundance (range 0.1%-96% of all BCR-ABL transcripts), but in only three samples abundance was higher than 15-20% - thus detectable also by conventional sequencing. Re-sequencing a set of samples in the same and independent runs confirmed the presence of the 35INS and demonstrated that this variant was not a PCR or sequencing artifact. Longitudinal analysis showed that the expression of 35INS fluctuated over time with no apparent correlation with response levels. In addition, DS detected one in-frame deletion in 20/41 (48%) CML patients and 7/16 (44%) Ph+ ALL patients, with an abundance ranging from 0.2% to 19%. This previously unreported variant consisted of a 72bp deletion (nt.1233-1304) at the junction of exon 6 to exon 7, that causes the loss of 24 residues (a.a. 359-383) of the KD. ConclusionsOur results further underline that DS technologies allow more accurate sequence characterization in comparison to conventional methods. Minor clones harboring insertions or deletions (always involving intron/exon junctions - which implicates alternative or aberrant splicing mechanisms) were found to be very frequent both in CML and in Ph+ ALL patients but, apparently, did not correlate with response or resistance to TKI therapy. In line with our findings, a very recent functional study has demonstrated that the truncated BCR-ABL protein resulting from the 35INS is kinase-inactive and should not play any role in TKI-resistance - in contrast to what had initially been hypothesized. However, further analysis of a larger number of samples would be needed to better understand the biological and clinical meaning of these minor clones surviving TKI therapy.Supported by PRIN 2009 (prot.2009JSMKY), Fondazione CARISBO, AIL, AIRC, FP7 ‘NGS-PTL', IGA NT 11555/13899. Disclosures:Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:NOVARTIS : Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Validation of a yeast functional assay for p53 mutations using clonal sequencing

We have previously tested biopsies from 1469 breast tumours with a p53 functional assay in the context of a prospective clinical trial (EORTC 10994/BIG 1-00). The goal of the trial was to determine whether p53 status could be used to select patients who would benefit from inclusion of taxanes in anthracycline-based chemotherapy. The results of the trial were negative. To test whether this was because the functional assay misclassified the tumours, we have reanalysed two groups of biopsies by Sanger sequencing and Roche 454 next generation sequencing (NGS). Comparison of yeast data with pooled cDNA sequencing data in an initial cohort of 69 biopsies showed that conventional sequencing is insensitive when the mutant p53 content is low. A second cohort of 48 biopsies was used to compare directly the yeast assay with Sanger and NGS technology. The mutant sequence was difficult to detect in sequence chromatograms of pooled cDNA, whereas NGS unequivocally identified mutations in every case classified as mutant by the functional assay. The NGS data showed that small deletions, probably caused by PCR splicing, account for most of the unexplained background in the yeast assay. We conclude that mutation detection techniques that test multiple clones, such as the p53 functional assay and NGS, are more reliable than Sanger sequencing of pooled DNA; that the high p53 mutation rate (44%) seen with the yeast assay in the EORTC 10994/BIG 1-00 trial reflects this high sensitivity; and that NGS with Roche 454 technology could be used to identify the p53 mutations in the remaining tumours previously tested in yeast in the EORTC10994/BIG 1-00 trial.

Exploring phylogenetic informativeness and nuclear copies of mitochondrial DNA (numts) in three commonly used mitochondrial genes: mitochondrial phylogeny of peppermint, cleaner, and semi-terrestrial shrimps (Caridea:Lysmata,Exhippolysmata, andMerguia)

Of paramount importance to studies that profit from molecular trees is the accuracy and robustness of the reconstructed phylogenies. Causes of systematic error that can mislead phylogenetic methods include nuclear copies of mitochondrial DNA (numts) and low phylogenetic informativeness (PI). Herein, numts and PI were explored in three mitochondrial genes commonly used for phylogenetic reconstruction: 16S, 12S, and cytochrome c oxidase I (COI). Shrimps from the genera Lysmata, Exhippolysmata, and Merguia were used as a model system. The existence of: (1) multiple bands on gels of COI and 12S polymerase chain reaction (PCR) products from various species; (2) double peaks, background noise, and ambiguity in sequence chromatograms of COI and 12S PCR products that produced a single clear band in other species; and (3) indels, stop codons, and considerable composition bias in COI-like cloned sequences of one problematic species (Lysmata seticaudata), was interpreted as evidence of pervasive non-functional nuclear copies of mitochondrial DNA (numts) of the targeted COI (and probably 12S) mtDNA fragment. The information content of the three mtDNA markers studied was investigated using PI profiling, spectral analysis, and neighbour-nets. Marker-specific PI profiles suggested that the COI marker has the highest information content and greatest power for resolving both shallow and deep nodes in trees depicting the phylogenetic relationship among the species studied. Nonetheless, spectral analysis of splits and neighbour-nets suggested that the 16S and 12S markers were equally or even more powerful than the COI marker for resolving nodes at all phylogenetic levels. Altogether, these analyses suggest that all three mtDNA markers are equally useful for resolving phylogenetic relationships in the shrimps studied, and that PI profiling is not necessarily useful to estimate overall gene utility. A ‘total-evidence’ phylogenetic analysis that included 34 species and used a concatenated data set of 1403 characters (from reliable 16S, 12S and COI sequences), demonstrated that the genus Lysmata is paraphyletic, and that the monophyletic clade comprising species of Lysmata and Exhippolysmata can be divided into four well-supported subclades (Neotropical, Cleaner, Cosmopolitan, and Morphovariable).

RECall for Automated Genotypic Tropism Testing

Standardization of sequence chromatogram analysis is required for consistent genotypic tropism determination across laboratories. A freely available, fast, and automated chromatogram analysis tool (RECall) provided tropism interpretations equivalent to those of manual sequence editing of 521 V3 loop HIV-1 population sequences, suggesting that RECall can be useful in standardizing genotypic tropism testing across laboratories.

New insights into character evolution, hybridization and diversity of Indian Nymphaea (Nymphaeaceae): evidence from molecular and morphological data

A comprehensive reassessment of Indian Nymphaea (Nymphaeaceae) based on morphology, RAPD and nucleotide sequence data of the ITS, trnK intron, matK and rbcL gene was conducted. Although considerable morphological variations have been reported, pollen colour and rhizome shape are two characters which have not been mentioned in previous studies. The transformation from yellow to white coloured pollen may have evolved independently and is probably associated with a strong selective pressure acted upon by the animal pollinators on white pollen of N. pubescens and N. rubra. Rhizome shape could easily discriminate among subgenera; the difference in shape (triangular, globular and cylindrical) probably highlights the success in their distribution. Our studies using RAPD revealed high genetic variability among individuals of N. caerulea. This may be attributed to the breeding system followed, which could be an outcrossing species. But the lack of genetic diversity in N. tetragona is probably due to founder events, whereby a solitary founder individual from China could have resulted in the establishment of this single population of ∼25–30 individuals with no detectable variations. Molecular cloning of the ITS region of N. rubra, necessitated by the presence of additional signals in the sequencing chromatogram, confirmed the origin of this plant taxon through hybridization involving N. lotus and N. pubescens as the parental species.

Direct Sequencing and RipSeq Interpretation as a Tool for Identification of Polymicrobial Infections

In this study, RipSeq Mixed, a software resolving uninterpretable mixed DNA sequencing chromatograms, revealed the bacterial content of 15 polymicrobial samples. Direct sequencing combined with RipSeq Mixed constitutes a valuable supplement to cultivation, particularly when cultivation is negative and direct sequencing is inconclusive despite continued clinical indications of infection.

Base-calling algorithm with vocabulary (BCV) method for analyzing population sequencing chromatograms.

Sanger sequencing is a common method of reading DNA sequences. It is less expensive than high-throughput methods, and it is appropriate for numerous applications including molecular diagnostics. However, sequencing mixtures of similar DNA of pathogens with this method is challenging. This is important because most clinical samples contain such mixtures, rather than pure single strains. The traditional solution is to sequence selected clones of PCR products, a complicated, time-consuming, and expensive procedure. Here, we propose the base-calling with vocabulary (BCV) method that computationally deciphers Sanger chromatograms obtained from mixed DNA samples. The inputs to the BCV algorithm are a chromatogram and a dictionary of sequences that are similar to those we expect to obtain. We apply the base-calling function on a test dataset of chromatograms without ambiguous positions, as well as one with 3–14% sequence degeneracy. Furthermore, we use BCV to assemble a consensus sequence for an HIV genome fragment in a sample containing a mixture of viral DNA variants and to determine the positions of the indels. Finally, we detect drug-resistant Mycobacterium tuberculosis strains carrying frameshift mutations mixed with wild-type bacteria in the pncA gene, and roughly characterize bacterial communities in clinical samples by direct 16S rRNA sequencing.

Subclonal evolution involving SF3B1 mutations in chronic lymphocytic leukemia

Subclonal evolution involving SF3B1 mutations in chronic lymphocytic leukemia

SeqTrace: A Graphical Tool for Rapidly Processing DNA Sequencing Chromatograms

Modern applications of Sanger DNA sequencing often require converting a large number of chromatogram trace files into high-quality DNA sequences for downstream analyses. Relatively few nonproprietary software tools are available to assist with this process. SeqTrace is a new, free, and open-source software application that is designed to automate the entire workflow by facilitating easy batch processing of large numbers of trace files. SeqTrace can identify, align, and compute consensus sequences from matching forward and reverse traces, filter low-quality base calls, and end-trim finished sequences. The software features a graphical interface that includes a full-featured chromatogram viewer and sequence editor. SeqTrace runs on most popular operating systems and is freely available, along with supporting documentation, at http://seqtrace.googlecode.com/.

Genetic Diversity as a Marker for Timing Infection in HIV-Infected Patients: Evaluation of a 6-Month Window and Comparison With BED

It has been reported that the increase in human immunodeficiency virus (HIV) sequence diversity in drug resistance surveillance specimens may be used to classify the duration of HIV infection as <1 or >1 year. We describe a mixed base classifier (MBC) optimized to categorize the duration of subtype B infections as <6 or >6 months on the basis of sequences for drug resistance surveillance specimens and compared MBC findings with those of serologic methods. The behavior of the MBC was examined across a range of thresholds for calling mixed bases. MBC performance was then evaluated using either complete pol sequences or sites reflecting evolutionary pressures (HLA selection sites, sites that increased in entropy over the course of infection, and codon positions). The MBC performance was optimal when secondary peaks on the sequencing chromatogram accounted for at least 15% of the area of primary peaks. A cutoff of <0.45% mixed bases in the pol region best identified recent infections (sensitivity = 82.7%, specificity = 78.8%), with improvement achieved by analyzing only sites that increased in entropy. In an extended data set of 1354 specimens classified by BED, the optimized MBC performed significantly better than a simple MBC (agreement, 68.98% vs 67.13%). If further validated, the MBC may prove beneficial for detecting recent infection and estimating the incidence of HIV infection.