In Silico Restriction Enzyme Digests to Minimize Mapping Bias in Genomic Sequencing

Roszik, Jason; Fenyőfalvi, György; Halász, László; Karányi, Zsolt; Székvölgyi, Lóránt

doi:10.1016/j.omtm.2017.06.003

Cited by 5 publications

(5 citation statements)

References 9 publications

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“…Previous studies (DaCosta & Sorenson, 2014; Kirschner et al, 2016; Roszik, Fenyőfalvi, Halász, Karányi, & Székvölgyi, 2017) also reported a bias caused by the restriction enzymes, especially toward first exons. Thus, the assumption of sequencing random fractions of the genome is not accomplished, and it depends on the restriction enzyme selected.…”

Section: Discussionmentioning

confidence: 90%

Evaluating restriction enzyme selection for genome reduction in conservation genomics

Lopez

Carreras

Pascual

et al. 2022

Preprint

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Conservation genomic studies in non-model organisms generally rely on genome reduction techniques based on restriction enzymes to identify population structure as well as candidate loci for local adaptation. These reduced libraries ensure a high density of SNP loci and high coverage for accurate genotyping. Despite the fraction of the genome that is sequenced is expected to be randomly located, the reduction of the genome might depend on the recognition site of the restriction enzyme used. Here, we evaluate the distribution and functional composition of loci obtained after Genotyping-by-sequencing (GBS) genome reduction with two widely used restriction enzymes (EcoT22I and ApeKI). To do so, we compared data from two endemic fish species (Symphodus ocellatus and Symphodus tinca, EcoT22I enzyme) and two ecosystem engineer sea urchins Paracentrotus lividus and Arbacia lixula, ApeKI enzyme). In brief, we mapped the sequenced loci to the phylogenetically closest reference genome available (Labrus bergylta for fish and Strongylocentrotus purpuratus for sea urchins), classified them as exonic, intronic, and intergenic, and studied their functionality by using GO terms. We detected an enrichment towards exonic or intergenic regions depending on the restriction enzyme used, and we did not detect differences between total loci and candidate loci for adaptation. Despite most GO terms being shared between species, the analysis of their abundance showed differences between taxonomic groups, which may be attributed to differences of the targeted loci. Our results highlight the importance of restriction enzyme selection and the need for high-quality annotated genomes in conservation genomic studies.

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Section: Discussionmentioning

confidence: 90%

Evaluating restriction enzyme selection for genome reduction in conservation genomics

Lopez

Carreras

Pascual

et al. 2022

Preprint

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“…Massive volumes of data are generated by high-throughput sequencing technology. Genotyping and mapping bias in genomic sequencing can be reduced using an In silico restriction enzyme [11]. In general, however, the analysis of In silico does not allow for evaluating the number of obtained DNA fragments of the given length to predict their visualization in the experiment [15].…”

Section: Resultsmentioning

confidence: 99%

“…Comparison of restriction patterns for two or more DNA sequences might be performed using In silico restriction digestion sites. When In silico restriction gene program comes into contact with a DNA sequence having a structure that matches part of the numerous restriction enzymes routinely utilized in laboratories to build recognition sites [11], it searches for restriction sites. Based on this information, gra1, rop1, and mic3 were selected as genetic markers for predicting genotype of T. gondii using In silico restriction site.…”

Section: Introductionmentioning

confidence: 99%

In silico restriction site analysis for characterization of Toxoplasma gondii isolate

Ekawasti

Cahyaningsih

Sa’diah

et al. 2022

IOP Conf. Ser.: Earth Environ. Sci.

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Toxoplasma gondii infection is a serious major public health concern. Toxoplasmosis is a disease that affects humans and warm-blooded animals all over the world. The virulence and severity of the sickness may be influenced by parasite load. The three clonal lineages for biological research are T. gondii genotypes I, II, and III. Using primer genes for gra1, rop1, and mic3, which have been identified as essential proteins for tachyzoite invasion and replication in host cells. PCR was used to determine the genotype of a T. gondii isolate from the Indonesian research center for veterinary science. In silico restriction site analysis was performed on T. gondii isolates using CLC sequence viewer 8.0 software to assess sequence data for the existence of restriction enzyme patterns. Predictable restriction fragment length polymorphism using In silico analysis. T. gondii sequencing genes are In silico digested with different restriction enzymes. The findings show that they can easily distinguish archetypal parasites from biotypes and estimate the genetic diversity of the parasites. According to the interpretation of the data, isolate T. gondii belongs to strain RH genotype I.

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“…Using in silico restriction digestion of given DNA sequences to predict the expected DNA fragments, the "digestDNA" function can be used to perform the digestion [45]. In silico analysis is an approach performed on a computer to analyze the restriction fragment length distribution.…”

Section: In Silico Restriction Gene Analysismentioning

confidence: 99%

“…A generally used genome fragmentation method, restriction endonuclease digestion may severely compromise genomic mapping resolution and prevent the practical annotation of certain chromosomal regions. In silico restriction enzyme digests to minimize mapping bias in genomic sequencing and result in proper DNA fragment size distributions and optimal resolution in genomic sequencing technologies [45]. In general, In silico analysis is predicts their visualization in the experiment.…”

Section: )mentioning

confidence: 99%

Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

et al. 2021

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Background and Aim: Toxoplasma gondii is a unicellular coccidian parasite distributed globally and is an important zoonotic pathogen. Approximately 30% of the human population worldwide is chronically infected with T. gondii. The pathogenicity of this species depends on the type originating from the clonal population. Techniques for more accurately determining the type of T. gondii have recently been developed using genetic markers. Specifically, T. gondii has been typed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This study aimed to identify sets of PCR-RFLP markers that have high power to discriminate genotyping of T. gondii and are easy to use and are easy to use. The objective of this study was to characterize virulent strain isolates of T. gondii by PCR-RFLP using 10 markers with DdeI. Materials and Methods: T. gondii tachyzoites (RH virulent strain) were derived from culture cells at the Indonesian Research Center for Veterinary Sciences. Genotyping was performed on T. gondii DNA extracted from cell cultured tachyzoites using 10 genetic markers of PCR-RFLP, namely, B1#1, B1#2, B1#3, SAG1#1, SAG1#2, P30, BAG1, ROP1, GRA1, and GRA7, with digestion using the restriction enzyme DdeI. Results: The 10 genes were amplified by PCR. Among them, three genetic markers, B1#3, ROP1, and GRA1, were genotyped by the PCR-RFLP using restriction enzyme DdeI. Overall, the findings showed that the specific RFLP profile of digestion of gene regions by DdeI could be used as a specific marker for the virulent biotype causative of toxoplasmosis. In addition, virulent strains of T. gondii can be easily detected by these markers. Conclusion: Three pairs of primers (B1#3, ROP1, and GRA1) with DdeI have proven useful for the diagnosis of acute toxoplasmosis (virulent strain biotype I). This proposed method is relatively simple, rapid, cheap, and can be performed in most laboratories, providing a practical approach for the routine analysis of T. gondii strains.

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In Silico Restriction Enzyme Digests to Minimize Mapping Bias in Genomic Sequencing

Cited by 5 publications

References 9 publications

Evaluating restriction enzyme selection for genome reduction in conservation genomics

Evaluating restriction enzyme selection for genome reduction in conservation genomics

In silico restriction site analysis for characterization of Toxoplasma gondii isolate

Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

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