Sequencing Of Individual Clones Research Articles

A highly accurate platform for clone-specific mutation discovery enables the study of active mutational processes.

Bulk whole genome sequencing (WGS) enables the analysis of tumor evolution but, because of depth limitations, can only identify old mutational events. The discovery of current mutational processes for predicting the tumor's evolutionary trajectory requires dense sequencing of individual clones or single cells. Such studies, however, are inherently problematic because of the discovery of excessive false positive (FP) mutations when sequencing picogram quantities of DNA. Data pooling to increase the confidence in the discovered mutations, moves the discovery back in the past to a common ancestor. Here we report a robust WGS and analysis pipeline (DigiPico/MutLX) that virtually eliminates all F results while retaining an excellent proportion of true positives. Using our method, we identified, for the first time, a hyper-mutation (kataegis) event in a group of ∼30 cancer cells from a recurrent ovarian carcinoma. This was unidentifiable from the bulk WGS data. Overall, we propose DigiPico/MutLX method as a powerful framework for the identification of clone-specific variants at an unprecedented accuracy.

Rapid Mapping of Protein Binding Sites and Conformational Epitopes by Coupling Yeast Surface Display to Chemical Labeling and Deep Sequencing.

Delineating the precise regions on an antigen that are targeted by antibodies is important for the development of vaccines and antibody therapeutics. X-ray crystallography and NMR are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, these are labor-intensive and require purified protein at high concentration. We have recently described [1] a rapid and reliable method that overcomes these constraints, using a panel of single cysteine mutants of the protein of interest and now provide protocols to facilitate its adoption. Mutants are displayed on the yeast cell surface either individually or as a pool, and labeled covalently with a cysteine specific probe. Binding site residues are inferred by monitoring loss of ligand or antibody binding by flow cytometry coupled to deep sequencing of sorted populations, or Sanger sequencing of individual clones. Buried cysteine residues are not labeled and library sizes are small, facilitating rapid identification of binding-site residues. The methodology was used to identify epitopes on the bacterial toxin CcdB targeted by twenty-four different monoclonal antibodies as well as by polyclonal sera. The method does not require purified protein or protein structural information and can be applied to a variety of display formats.

A 3' RACE protocol to confirm polyadenylation sites.

A routine procedure in the study of polyadenylation involves the determinations of the junctions of the mRNA body and the poly(A) tail (the poly(A) site). This is typically accomplished by selectively amplifying the 3'-ends of cDNAs (3'-Rapid Amplification of cDNA Ends, or 3'-RACE), followed by sequencing of individual clones. We have developed a modification of the standard 3'-RACE protocol that couples high specificity with the ability to characterize thousands (or more) of individual sequences. This protocol may be used for numerous purposes, including the confirmation of results obtained using high throughput sequencing technologies.

Abstract LB-218: Molecular changes associated with acquired resistance to crizotinib in ROS1-rearranged non-small cell lung cancer (NSCLC)

Abstract Background: Although ROS1-rearranged NSCLC is sensitive to crizotinib, a dual inhibitor of c-MET and ALK, an acquired resistance is inevitable. Recently, ROS1 G2032R mutation was identified in a patient with CD74-ROS1 rearranged NSCLC who developed an acquired resistance to crizotinib. Here, we identified novel molecular changes in crizotinib-resistant tumors from a NSCLC patient with CD74-ROS1 rearrangement and in HCC78 cells harboring SLC34A2-ROS1 fusion that showed crizotinib resistance (HCC78CR1, CR2 and CR3). Methods: HCC78CR1, -2 and -3 cells were generated by increasing dose of crizotinib to 1 μM. ROS1 kinase domain mutations were examined in fresh tumor tissues from a patient and in HCC78CR1, -2 and -3 cells by direct sequencing of individual clones. Whole-transcriptome sequencing (RNA-seq) was performed to identify up-regulated pathway in HCC78CR3 compared with HCC78. Cell proliferation, phosphorylation of RTKs and ROS1 downstream signaling pathways were compared among HCC78 and HCC78CR1, -2, -3 cell lines. BaF3 cells that constantly express ROS1 variants were constructed to evaluate a crizotinib resistance. Results: ROS1 G2032R in crizotinib-resistant tumor from a patient and L2155S from HCC78CR1, -2 cells were identified by direct sequencing of individual clones. ROS1 G2032R mutation was identified in 100% of cDNA clones of crizotinib-resistant tumors from a patient. However, this was not found in normal tissues and crizotinib-pretreatment tumor tissues. cDNA clones of HCC78CR1 and 2 cells harbored ROS1 L2155S mutations at a frequency of 73.3% and 76.2% respectively. Each BaF3 cells expressing ROS1 G2032R or L2155S mutation exhibited 128 and 10 fold higher IC50 value to crizotinib than those expressing wild-type CD74-ROS1, respectively. Downstream signaling molecules such as ERK and SHP2 were more activated in HCC78CR1, -2 and -3 cells than in HCC78 cells. In addition, HCC78CR1 and -2 cells displayed a spindle cell-like morphology with decreased E-cadherin and increased vimentin and fibronectin expression, which was not observed in HCC78 cells. RNA-seq and qRT-PCR demonstrated that EGFR mRNA expression levels were 2.6 and 5.1-fold up-regulated in HCC78CR3 cells than in HCC78 cells, respectively. EGFR signaling antibody array also revealed that EGFR pathway is significantly up-regulated in HCC78CR3 cells than in HCC78 cells. Conclusions: ROS1 G2032R and L2155S mutations are associated with acquired resistance to crizotinib in ROS1-rearranged NSCLC. In addition, epithelial-to-mesenchymal transition-like changes and EGFR activation may confer an acquired resistance to crizotinib. Citation Format: Ahn ah Song. Molecular changes associated with acquired resistance to crizotinib in ROS1-rearranged non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-218. doi:10.1158/1538-7445.AM2014-LB-218

Expression of Dual-Specificity Phosphatase 5 Pseudogene 1 (DUSP5P1) in Tumor Cells

Sequencing of individual clones from a newly established cDNA library from the chemoresistant Hodgkin's lymphoma cell line L-1236 led to the isolation of a cDNA clone corresponding to a short sequence from chromosome 1. Reverse transcriptase-polymerase chain reaction indicated high expression of this sequence in Hodgkin's lymphoma derived cell lines but not in normal blood cells. Further characterization of this sequence and the surrounding genomic DNA revealed that this sequence is part of a human endogenous retrovirus locus. The sequence of this endogenous retrovirus is interrupted by a pseudogene of the dual specificity phosphatase 5 (DUSP5). Reverse transcriptase-polymerase chain reaction revealed high expression of this pseudogene (DUSP5P1) in HL cell lines but not in normal blood cells or Epstein-Barr virus-immortalized B cells. Cells from other tumor types (Burkitt's lymphoma, leukemia, neuroblastoma, Ewing sarcoma) also showed a higher DUSP5P1/DUSP5 ratio than normal cells. Furthermore, we observed that higher expression of DUSP5 in relation to DUSP5P1 correlated with the expression of the pro-apoptotic factor B cell leukemia/lymphoma 2-like 11 (BCL2L11) in peripheral blood cells and HL cells. Knock-down of DUSP5 in HL cells resulted in down-regulation of BCL2L11. Thus, the DUSP5/DUSP5P1 system could be responsible for regulation of BCL2L11 leading to inhibition of apoptosis in these tumor cells.

Approaches to Enzyme and Substrate Design of the Murine Dnmt3a DNA Methyltransferase

Dnmt3a-C, the catalytic domain of the Dnmt3a DNA-(cytosine-C5)-methyltransferase, is active in an isolated form but, like the full-length Dnmt3a, shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a-C methylated its own expression plasmid in E. coli, and protected it from cleavage by methylation-sensitive restriction enzymes. However, despite screening about 400 clones that were selected in three rounds from a random mutagenesis library of 60 000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids that had lost the restriction sites. To improve the catalytic activity of Dnmt3a-C by optimization of the sequence of the DNA substrate, we analyzed its flanking-sequence preference in detail by bisulfite DNA-methylation analysis and sequencing of individual clones. Based on the enrichment and depletion of certain bases in the positions flanking >1300 methylated CpG sites, we were able to define a sequence-preference profile for Dnmt3a-C from the -6 to the +6 position of the flanking sequence. This revealed preferences for T over a purine at position -2, A over G at -1, a pyrimidine at +1, and A and T over G at +3. We designed one "good" substrate optimized for methylation and one "bad" substrate designed not to be efficiently methylated, and showed that the optimized substrate is methylated >20 times more rapidly at its central CpG site. The optimized Dnmt3a-C substrate can be applied in enzymatic high-throughput assays with Dnmt3a-C (e.g., for inhibitor screening), because the increased activity provides an improved dynamic range and better signal/noise ratio.

Bacterial Diversity in <I>Amblyomma americanum</I> (Acari: Ixodidae) With a Focus on Members of the Genus <I>Rickettsia</I>

The lone star tick, Amblyomma americanum (Acari: Ixodidae), is commonly reported from people and animals throughout the eastern U.S. and is associated with transmission of a number of emerging diseases. To better define the microbial communities within lone star ticks, 16S rRNA gene based analysis using bacteria-wide primers, followed by sequencing of individual clones (n = 449) was used to identify the most common bacterial operational taxonomic units (OTUs) present within colony-reared and wild A. americanum. The colony-reared ticks contained primarily sequence affiliated with members of the genus Coxiella (89%; 81/91), common endosymbionts of ticks, and Brevibacterium (11%; 10/91). Similarly, analysis of clones from unfed wild lone star ticks revealed that 96.7% (89/92) of all the OTUs identified were affiliated with Coxiella-like endosymbionts, as compared with only 5.1-11.7% (5/98-9/77) of those identified from wild lone star ticks after feeding. In contrast, the proportion of OTUs identified as Rickettsia sp. in wild-caught ticks increased from 2.2% (2/92) before feeding to as high as 46.8% (36/77) after feeding, and all Rickettsia spp. sequences recovered were most similar to those described from the spotted fever group Rickettsia, specifically R. amblyommii and R. massiliae. Additional characterization of the Rickettsiales tick community by polymerase chain reaction, cloning, and sequencing of 17 kDa and gltA genes confirmed these initial findings and suggested that novel Rickettsia spp. are likely present in these ticks. These data provide insight into the overall, as well as the rickettsial community of wild lone star ticks and may ultimately aid in identification of novel pathogens transmitted by A. americanum.

Effect of Alcohol Consumption on CpG Methylation in the Differentially Methylated Regions of H19 and IG‐DMR in Male Gametes—Implications for Fetal Alcohol Spectrum Disorders

Exposure to alcohol in utero is the main attributable cause of fetal alcohol spectrum disorders (FASD) which in its most severe form is characterized by irreversible behavioral and cognitive disability. Paternal preconception drinking is not considered to be a significant risk factor, even though animal studies have demonstrated that chronic paternal alcohol consumption has a detrimental effect on the physical and mental development of offspring even in the absence of in utero alcohol exposure. It has been documented that alcohol can reduce the levels and activity of DNA methyltransferases resulting in DNA hypomethylation and that reduced methyltransferase activity can cause activation of normally silenced genes. The aim of this study was to establish a link between alcohol use in men and hypomethylation of paternally imprinted loci in sperm DNA in genomic regions critical for embryonic development, thus providing a mechanism for paternal effects in the aetiology of FASD. Sperm DNA from male volunteers was bisulfite treated and the methylation patterns of 2 differentially methylated regions (DMRs), H19 and IG-DMR, analyzed following sequencing of individual clones. The methylation patterns were correlated with the alcohol consumption levels of the volunteer males. There was a pattern of increased demethylation with alcohol consumption at the 2 imprinted loci with a significant difference observed at the IG-DMR between the nondrinking and heavy alcohol consuming groups. Greater inter-individual variation in average methylation was observed at the H19 DMR and individual clones were more extensively demethylated than those of the IG-DMR. CpG site #4 in the IG-DMR was preferentially demethylated among all individuals and along with the H19 DMR CpG site #7 located within the CTCF binding site 6 showed significant demethylation in the alcohol consuming groups compared with the control group. This study demonstrates a correlation between chronic alcohol use and demethylation of normally hypermethylated imprinted regions in sperm DNA. We hypothesize that, should these epigenetic changes in imprinted genes be transmitted through fertilization, they would alter the critical gene expression dosages required for normal prenatal development resulting in offspring with features of FASD.

TaqMan-based, real-time quantitative polymerase chain reaction method for RNA editing analysis

Abnormal adenosine to inosine (A-to-I) messenger RNA (mRNA) editing has been linked to several disease states afflicting the central nervous system. Here we report an assay to determine RNA editing frequencies at specific sites that is based on quantitative polymerase chain reaction (qPCR) with TaqMan probes. The assay was tested by measuring the frequency of the A-to-I mRNA editing at the Q/R site of the human kainate receptor subunit GluR5 and was compared with two established methods of assessing RNA editing: sequencing of individual clones and restriction analysis. The qPCR assay displayed high sensitivity and reproducibility, demonstrated exceptional discrimination between edited and unedited transcript variants, and proved to have several advantages over the other editing methods. Due to the fact that TaqMan-based qPCR technology can be easily adapted to different editing targets, the increased capabilities afforded by this new technique should facilitate various RNA editing studies that aim to elucidate the role of this process in normal physiology and in disease.

Rapid detection of mutations in the human-derived Pneumocystis carinii dihydropteroate synthase gene associated with sulfa resistance.

Recent studies have shown that point mutations in the dihydropteroate synthase (DHPS) gene of human-derived Pneumocystis carinii are related to exposure to sulfa drugs and possibly represent the emergence of sulfa resistance. We developed a simple single-strand conformation polymorphism (SSCP) method to permit rapid detection of these mutations. With plasmid constructs, SSCP was able to detect as little as 10% of a minority population. The SSCP assay was compared to direct sequencing for typing the DHPS gene by examining 37 clinical isolates with known DHPS sequences and 41 clinical isolates with unknown DHPS sequences. The typing results were consistent between these two methods for all isolates except 11 in which mutations were detected by SSCP but not by direct sequencing. Sequencing of individual clones after subcloning confirmed the presence of mutations in a minority population as determined by SSCP. SSCP is a very simple and sensitive method for rapid identification of P. camii DHPS mutations.

The FXYD Gene Family of Small Ion Transport Regulators or Channels: cDNA Sequence, Protein Signature Sequence, and Expression

A gene family of small membrane proteins, represented by phospholemman and the γ subunit of Na,K-ATPase, was defined and characterized by the analysis of more than 1000 related ESTs (expressed sequence tags). In addition to new and more complete cDNA sequence for known family members (including MAT-8, CHIF, and RIC), the findings included two new family members and new splicing variants. A large number of EST replicates made it possible to derive curated DNA sequence with higher confidence and accuracy than from the sequencing of individual clones. The family has a core motif of 35 invariant and conserved amino acids centered on a single transmembrane span. Features of each predicted protein product were compared, and tissue distributions were determined. The gene family was named FXYD (pronounced fix-id) in recognition of invariant amino acids in its signature motif. The abundant proteins are involved in the control of ion transport.

Pathological exon skipping in an HNPCC proband withMLH1 splice acceptor site mutation

One of the most commonly mutated mismatch repair genes in human nonpolyposis colorectal cancer (HNPCC) is MLH1. We identified a splice site mutation in MLH1 in a colorectal cancer proband (T-to-A at position -11 of intron 1 splice acceptor) and investigated its functional consequences by RT-PCR, using lymphocyte mRNA from the proband, two noncarrying siblings, and one unrelated individual. Subcloning of PCR products followed by sequencing of individual clones revealed increased transcript heterogeneity in the mutation carrier, attributable to the presence of a variety of mRNA forms lacking exon 2, or combinations of exons 2, 4, 6, 9, and 10. The full-length transcript subcloned from the mutation carrier was detected with a much reduced frequency, suggesting that only the wild-type allele produced functional MLH1 mRNA. The three noncarriers expressed some previously described transcripts and several novel variants, but none that lacked exon 2. The results are consistent with the hypothesis that this splice site mutation causes skipping of MLH1 exon 2 in a large proportion of mRNA transcripts derived from the mutated allele. Such an observation strengthens the case for identifying the mutation as pathogenic in this HNPCC family, which is of interest given the rarity of exon skipping defects resulting from splice acceptor site mutations outside the invariant AG dinucleotide.

Inherited susceptibility to aminoglycoside ototoxicity: Genetic heterogeneity and clinical implications

Purpose: Aminoglycoside-induced ototoxicity appears to have a genetic susceptibility in some individuals, and theA1555G mutation in the mitochondrial 12S ribosomal RNA gene has been shown to be responsible for this susceptibility in all familial cases. An Italian family with 5 family members who became deaf after aminoglycoside exposure presented to us, and molecular analysis excluded the Al 555G mutation. The purpose of this study is to identify the molecular basis for the aminoglycoside susceptibility in this family. Patients and Methods: Two sisters and three of their children developed severe to profound high-frequency hearing loss after aminoglycoside exposure. DNA was extracted from the blood of these individuals and their unaffected relatives, and analyzed for mitochondrial DNA mutations. The region around nucleotide 961 was also cloned and individual clones were sequenced. Results: Sequencing of the 12S ribosomal RNA gene revealed a thymidine deletion at position 961, with a complex pattern of sequence around this mutation. Sequencing of individual clones around the 961 mutation demonstrated a varying number of inserted cytosines in different mitochondrial molecules. Conclusion: This family establishes the nucleotide 961 thymidine deletion associated with a varying number of inserted cytosines in the mitochondrial 12S ribosomal RNA gene as the second pathogenic mutation that can predispose to aminoglycoside ototoxicity. It demonstrates the clinical relevance of taking a family history before administering aminoglycosides to any patient. In addition, it would be desirable for sporadic patients with aminoglycoside-induced hearing loss to be screened with molecular tests for the presence of the 1555 and 961 mutations. Such screening could significantly decrease the prevalence of aminoglycoside-induced hearing loss.