Genomic Sequencing Procedure Research Articles

The History and Impact of Molecular Coding Changes on Coverage and Reimbursement of Molecular Diagnostic Tests: Transition from Stacking Codes to the Current Molecular Code Set Including Genomic Sequencing Procedures

Changes in coding and coverage generate an uncertain reimbursement environment for molecular pathology laboratories. We analyzed our experience with two representative molecular oncology tests: a T-cell receptor (TCR) β rearrangement test and a large (467-gene) cancer next-generation sequencing panel, the Columbia Combined Cancer Panel (CCCP). Before 2013, the TCR β test was coded using stacked current procedural terminology codes and subsequently transitioned to a tier 1 code. CCCP was coded using a combination of tier 1 and 2 codes until 2015, when a new Genomic Sequencing Procedure code was adopted. A decrease in reimbursement of 61% was observed for the TCR β test on moving from stacking to tier 1 codes. No initial increase in total rejection rate was observed, but a subsequent increase in rejection rates in 2015 and 2016 was noted. The CCCP test showed a similar decrease (48%) in reimbursement after adoption of the new Genomic Sequencing Procedure code and was accompanied by a sharp increase in rejection rates both on implementation of the new code and over time. Changes in coding can result in substantial decreases in reimbursement. This may be a barrier to patient access because of the high cost of molecular diagnostics. Revisions to the molecular code set will continue. These findings help laboratories and manufacturers prepare for the financial impact and advocate appropriately.

Neurodegeneration with brain iron accumulation: update on pathogenic mechanisms.

Perturbation of iron distribution is observed in many neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease, but the comprehension of the metal role in the development and progression of such disorders is still very limited. The combination of more powerful brain imaging techniques and faster genomic DNA sequencing procedures has allowed the description of a set of genetic disorders characterized by a constant and often early accumulation of iron in specific brain regions and the identification of the associated genes; these disorders are now collectively included in the category of neurodegeneration with brain iron accumulation (NBIA). So far 10 different genetic forms have been described but this number is likely to increase in short time. Two forms are linked to mutations in genes directly involved in iron metabolism: neuroferritinopathy, associated to mutations in the FTL gene and aceruloplasminemia, where the ceruloplasmin gene product is defective. In the other forms the connection with iron metabolism is not evident at all and the genetic data let infer the involvement of other pathways: Pank2, Pla2G6, C19orf12, COASY, and FA2H genes seem to be related to lipid metabolism and to mitochondria functioning, WDR45 and ATP13A2 genes are implicated in lysosomal and autophagosome activity, while the C2orf37 gene encodes a nucleolar protein of unknown function. There is much hope in the scientific community that the study of the NBIA forms may provide important insight as to the link between brain iron metabolism and neurodegenerative mechanisms and eventually pave the way for new therapeutic avenues also for the more common neurodegenerative disorders. In this work, we will review the most recent findings in the molecular mechanisms underlining the most common forms of NBIA and analyze their possible link with brain iron metabolism.

Lineage specific evolutionary events on SFTPB gene: Alu recombination-mediated deletion (ARMD), exonization, and alternative splicing events

SFTPB gene encoding pulmonary surfactant protein has been investigated to have transcript variants related with transposable elements (TEs). To investigate the alternative splicing event on SFTPB gene, various primate samples (tissue RNA and genomic DNA) were used. Two different transcript variants (T1-2 and T2-2) were newly identified by RT-PCR analysis. T1-1,-2 and T2-1,-2 are TE-related and original transcripts, respectively. T1-1 transcript was investigated to be a lung and human specific alternative transcript. T2-1 transcript shows dominant expression pattern in lung tissues. T1-related TEs (LTR7B and AluSx) were investigated by genomic PCR and sequencing procedure of different primate genomic DNAs. Sequencing analysis indicated that they had been integrated into our common ancestor genome before the divergence of simian and prosimian and Alu recombination-mediated deletion (ARMD) event had been occurred on our common ancestor genome after the divergence of New World monkeys and Old World monkeys in SFTPB gene 3′ UTR region. Taken together, integration event of LTR7B and AluSx on SFTPB gene seem to cause lineage specific events via general exonization event (New World monkeys and prosimian), ARMD event (Old World monkeys and hominoids), and alternative splicing (human) during the primate evolution.

Accelerated bisulfite-deamination of cytosine in the genomic sequencing procedure for DNA methylation analysis

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The determination is usually performed using sodium bisulfite solutions of 3-5 M concentration with an incubation period of 12-16 hr at 50 degrees C. We demonstrate here that this deamination can be speeded up significantly by increasing the bisulfite concentration and the temperature with which the reaction is performed. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at pH 5.4 and 90 degrees C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer an improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation.

DNA methylation analysis: speedup of bisulfite-mediated deamination of cytosine in the genomic sequencing procedure.

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The deamination is usually performed using sodium bisulfite solutions of 3-5 M concentration with an incubation period of 12-16 hr at 50 °C. We demonstrate here that this deamination can be speeded up significantly. We prepared a solution of 10 M bisulfite concentration of pH 5.4 and used it to treat DNA at temperatures up to 90 °C. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at 90 °C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer a significantly improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation. (Communicated by Takashi SUGIMURA, M.J.A.)

High-Speed Conversion of Cytosine to Uracil in Bisulfite Genomic Sequencing Analysis of DNA Methylation

Bisulfite genomic sequencing is a widely used technique for analyzing cytosine-methylation of DNA. By treating DNA with bisulfite, cytosine residues are deaminated to uracil, while leaving 5-methylcytosine largely intact. Subsequent PCR and nucleotide sequence analysis permit unequivocal determination of the methylation status at cytosine residues. A major caveat associated with the currently practiced procedure is that it takes 16-20 hr for completion of the conversion of cytosine to uracil. Here we report that a complete deamination of cytosine to uracil can be achieved in shorter periods by using a highly concentrated bisulfite solution at an elevated temperature. Time course experiments demonstrated that treating DNA with 9 M bisulfite for 20 min at 90 degrees C or 40 min at 70 degrees C all cytosine residues in the DNA were converted to uracil. Under these conditions, the majority of 5-methylcytosines remained intact. When a high molecular weight DNA derived from a cell line (containing a number of genes whose methylation status was known) was treated with bisulfite under the above conditions and amplified and sequenced, the results obtained were consistent with those reported in the literature. Although some degradation of DNA occurred during this process, the amount of treated DNA required for the amplification was nearly equal to that required for the conventional bisulfite genomic sequencing procedure. The increased speed of DNA methylation analysis with this novel procedure is expected to advance various aspects of DNA sciences.

DNA methylation and epigenetic inheritance

Mammalian cell lines silence genes at low frequency by the methylation of promoter sequences. These silent genes can be reactivated at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). The inactive and active epigenetic states of such genes are stably inherited. A method for silencing genes is now available. It involves treatment of permeabilized cells with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) which is incorporated into DNA. The methylation of promoter sequences has been confirmed using the bisulfite genomic sequencing procedure. Methylated oligonucleotides homologous to promoter sequences might be used to specifically target and silence given genes, but results so far have not been conclusive. Treatments that silence or reactivate genes by changing DNA methylation can be referred to as epimutagens, as distinct from mutagens that act by changing DNA sequences. The epimutagen 5-aza-CR reactivates genes but has little mutagenic activity, whereas standard mutagens (such as ethyl methane sulfonate and ultraviolet light) have little reactivation activity. Nevertheless, much more information is required about the effects of DNA-damaging agents in changing DNA methylation and gene activity and also about the role of epimutations in tumor progression.

Identification of 5-Methylcytosine in Complex Genomes

Cytosine methylation is attracting new attention for regulatory roles in gene expression and there is an increasing interest in detecting, at a single-base resolution, any 5-methylcytosine in genes from complex genomes. Differential base modification by chemicals followed by PCR-based genomic sequencing procedures can provide the resolution, sensitivity, and specificity required for such a goal. The various methods available are not devoid of artifacts but if used carefully and in combination, very reliable information can be obtained. We compare the methods using bisulfite and conventional PCR with those using either hydrazine or potassium permanganate and ligation-mediated PCR and provide a step-by-step description of the corresponding procedures.

Rapid tamoxifen-induced inactivation of an estrogenic response is accompanied by a localized epigenetic modification but not by mutations.

In a previous study (Cancer Res 54: 5860-5866, 1994), we observed irreversible inactivation of a chimeric estrogenic response induced by the antiestrogen 4-hydroxytamoxifen. This rapidly occurring effect (t1/2= 7 days) was not a consequence of a cell selection process, nor of a loss of estrogen receptor functionality, but was a direct antiestrogen effect occurring on every cell at the transcriptional level. In the present study, we analyzed the detailed methylation status of the chimeric gene, and investigated the gene for the presence of mutations. The inactivation process was found to be strictly correlated with a modification at a methylation-sensitive restriction site Not I borne by the integrated gene. As the gene promoter contains part of the Herpes simplex virus promoter for thymidine kinase. which is a CpG-rich promoter, we investigated the CpGs located in this part of the promoter by genomic sequencing procedures. None of these CpGs were methylated, suggesting that the inactivation process was not driven by particular modifications of this foreign part of the promoter. Furthermore, no mutations were found in the entire gene promoter of inactivated cells. In conclusion, the present study highlighted a connection between the rapid silencing of an estrogenic response induced by 4-hydroxytamoxifen, and a localized epigenetic modification of the corresponding gene. No genotoxicity of 4-hydroxytamoxifen was observed. Similar epigenetic modifications might also occur for natural genes, and lead to the acquisition of a new cell phenotype.

A method to estimate the percent loss of cytosine methyl groups at defined CpG sites in liver DNA from methyl-deficient rats

Dietary methyl deficiency provides an ideal in vivo model system in which to study progressive alterations in DNA methylation patterns as they occur during multistage hepatocarcinogenesis. Weanling male F344 rats were given a semipurified diet deficient in the methyl-donors choline, methionine, and folic acid for a 36-week period with sampling intervals at 3, 9, 24, and 36 weeks. Using a genomic sequencing procedure based on the PCR amplification of bisulfite-modified DNA, the methylation status of individual CpG sites within exons 6 and 7 of the p53 gene in liver samples from control and deficient rats was assessed. Treatment of denatured nuclear DNA with sodium bisulfite converts unmethylated cytosine residues to uracil, which are then amplified as thymine in the PCR reaction. In contrast, methylated cytosines are resistant to bisulfite deamination under these reaction conditions and are amplified as cytosine. In this report, we describe a novel application of automated sequencing technology to estimate the proportion of methylated cytosines present at defined CpG sites within the total population of DNA molecules extracted. Using the bisulfite conversion-PCR genomic sequencing method, we demonstrate the validity of peak height analysis of co-eluting peaks in the autosequencer electrophoregram to estimate the percent methylation at a defined CpG site. The sensitivity of this method is demonstrated by the progressive loss of methyl groups at a defined CpG site in the methyl-deficient rats after 9, 24, and 36 weeks. The application of this sequence-specific technology will allow site-specific definition of the methylation status of each CpG site within a coding sequence or promoter region and should provide new insights into mechanisms and consequences of methylation dysregulation as a result of dietary deprivation of methyl donors.

Genomic sequencing by ligation-mediated PCR.

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited due to the complexity of the mammalian genome. Ligation-mediated PCR (LMPCR) is a sensitive genomic sequencing procedure that generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. This genomic sequencing procedure can be adapted for various methylation, in vivo footprinting and DNA adduct mapping procedures. We provide a detailed protocol for genomic sequencing by LMPCR and discuss the principles and applications of the method.

39] Mapping DNA-protein interactions in cells and nuclei: Genomic sequencing by template purification

Publisher Summary This chapter presents a procedure for analyzing DNA-protein interactions on genomic DNA using template purification and genomic sequencing. The principles of the purification method and genomic sequencing are discussed in the chapter. Precise protocols for the treatment of cells with dimethyl sulfate (DMS) or KMnO4, and the isolation of nuclei for DNase I footprinting are presented. A number of genomic sequencing procedures, principally aimed at determining at the nucleotide level DNA-protein interactions on genomic DNA, have been developed. Although these sequencing procedures depend on different approaches, they all are difficult to carry out. The principal limitation is that a sequencing signal must be generated from a single-copy sequence which, for man, or mouse, is present in a total of about 3 × 109 bp of genomic DNA. Finally, protocols for the purification of a specific sequence from whole genomic DNA followed by detection of a sequence ladder using primer extension are also described.

Differential sensitivity to loss of cytosine methyl groups within the hepatic p53 gene of folate/methyl deficient rats.

Dietary folate/methyl deficiency provides a unique model of endogenous hepatocarcinogenesis in which to study progressive alterations in DNA methylation patterns during tumor progression in vivo. Weanling male F344 rats were given a semi-purified diet deficient in the methyl donors choline, methionine and folic acid for a period of 9 weeks. Using a genomic sequencing procedure based on the PCR amplification of bisulfite-modified DNA, the methylation status of individual CpG sites within exons 6 and 7 of the p53 gene in liver samples from control and deficient rats was determined. Treatment of denatured nuclear DNA with sodium bisulfite quantitatively converts all cytosine residues to uracil which are then amplified as thymine in the PCR reaction. In contrast, 5-methylcytosine is resistant to bisulfite deamination under the reaction conditions and is amplified as cytosine. Automated sequencing of bisulfite-modified DNA will then elucidate the methylation status of each cytosine residue within a defined gene sequence. In addition to evaluation of the methylation status of the p53 gene, the relative activity of the DNA methyltransferase was also quantified in nuclear extracts from control and folate/methyl deficient rats. The results indicate that specific 5-methyl cytosines within the hepatic p53 gene from methyl deficient rats are resistant to demethylation despite the diet-induced decrease in S-adenosylmethionine and the increase in cell proliferation associated with this dietary intervention. Progressive demethylation was observed at other methylated cytosine residues in folate/methyl deficient rats after 9 weeks despite a paradoxical increase in DNA methyltransferase activity. The application of this sequence-specific technology will allow the definition of the methylation status of every CpG site within a coding sequence or promoter region and should provide new insights into mechanisms and consequences of methylation dysregulation during progressive multistage carcinogenesis.

Genomic sequencing by ligation mediated polymerase chain reaction using direct blotting and non-radioactive detection.

Genomic sequencing has become an important tool for analyzing uncloned cellular DNA with regard to the methylation status of cytidines as well as to DNA-protein interactions within cells. The hybridization step of the genomic sequencing procedure requires a very high sensitivity, rendering the method fairly difficult. Using a modified ligation mediated polymerase chain reaction procedure (LMPCR) and a sensitive non-radioactive detection method, we have developed a procedure avoiding the high amounts of radioactivity formerly needed for detection of chemically cleaved genomic DNA. The detection limit of our method of genomic sequencing is less than 1 microgram mammalian DNA, which is much better than the detection limit of the original genomic sequencing method and comparable with the detection limit of radioactive detection after the LMPCR procedure. In addition to the advantages of the non-radioactive detection technique we simplified the blotting step of the genomic sequencing procedure by using the direct blotting electrophoresis method. The method was applied to a region 5' to the human c-myc promoter of HeLa cells and was able to verify the sequence obtained by other authors and to specify the methylation status of five CpG-pairs within this sequence.

Genomic sequencing and methylation analysis by ligation mediated PCR.

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.

Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli.

The genomic sequencing procedure is applied to the direct sequencing of uncharacterized regions of bacterial DNA by a "multiplex walking" approach. Samples of bulk Escherichia coli DNA are cut with various restriction enzymes, subjected to chemical sequencing degradations, run in a sequencing gel, and transferred to nylon membranes. When a labeled oligomer is hybridized to a membrane, a sequence ladder appears wherever the probe lies near a restriction cut. New probes, based on sequence that lies beyond other restriction sites, are then synthesized, and the membranes are reprobed to reveal new sequence. Repeated cycles of oligomer probe synthesis and subsequent reprobing permit rapid sequence walking along the genome. This oligomer walking technique was used to sequence the pyruvate kinase (EC 2.7.1.40) gene in E. coli without resorting to cloning or to library construction. The sequenced region was amplified by the polymerase chain reaction and subsequently transcribed and translated using both in vivo and in vitro systems, and the resultant gene product characterized to show that the gene encodes the type I isoform of pyruvate kinase.

Genomic sequencing.

Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.