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FAQ

NGS sequencing

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Common Issues in Whole Genome de novo Sequencing

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How to Ensure the Reliability of Assembly Results?To ensure the reliability of assembly results, in addition to maintaining Contig N50 and Scaffold N50, it's crucial to assess assembly quality. This can be achieved through various methods:1. **EST and RNA Data:** - Evaluate the integrity of assembled genes using EST (Expressed Sequence Tag) and RNA data.2. **BAC Data:** - Check for misassemblies or misjoins using BAC (Bacterial Artificial Chromosome) data.3. **Conserved Gene Evaluation:** - Assess genome assembly completeness through methods like CEGAM or BUSCO, which evaluate the pr…

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Whole Genome Bisulfite Sequencing (WGBS) Common Issues

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Translation:**Which species can be studied using WGBS?**To conduct whole-genome methylation analysis, the species should meet the following criteria:a. The species must be eukaryotic.b. The species should have a reference genome, assembled at least to the scaffold level.c. It should have relatively comprehensive annotations.**What are the advantages of Whole Genome Bisulfite Sequencing (WGBS)?**At the whole-genome level, WGBS detects methylation sites at single-base resolution. It not only accurately identifies changes in methylation levels in common regions such as CpG islands but also analyz…

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Common Issues in Small RNA Sequencing

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Translation:**Can Small RNA Sequencing be conducted for Species without a Reference Genome?**Small RNA sequencing studies can be conducted for species without a reference genome. However, in such cases, it is necessary to perform de novo transcriptome assembly to generate a transcriptome, which can then serve as a reference sequence for small RNA sequencing analysis.**What does the Preference Analysis Graph of the First and Each Base in Sample miRNA Analysis Indicate?**In miRNA precursor development into mature forms, the process is orchestrated by Dicer enzyme cleavage. The specificity of cle…

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Common Issues in circRNA Sequencing

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**Differences between Chip Technology and Second-Generation Sequencing Technology in Analyzing circRNA:**Chip technology can only detect known circRNAs, and the noise signal is relatively large. Currently, research on circRNAs is relatively limited, and circRNA annotation is not comprehensive. Additionally, circRNA expression has strong temporal and spatial specificity, making it challenging to ensure that the data obtained under experimental conditions match the data in the database. This may result in the loss of a significant number of specifically expressed circRNAs. Second-generation sequ…

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Common Issues in lncRNA Sequencing

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**Which Species Can be Studied for lncRNA Research?**To conduct lncRNA analysis, the species must meet the following criteria:a. The species must be eukaryotic.b. The species must have a reference genome, at least assembled to the scaffold level.c. The species should have relatively comprehensive annotations.**Why Remove rRNA During lncRNA Sequencing Library Construction?**In lncRNA, only the 3' end of lincRNA carries a polyA structure, while other lncRNAs lack a polyA structure. Therefore, to obtain a more comprehensive understanding of lncRNA information, it is not recommended to use oli…

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Common Issues in 16S, 18S, ITS, and Other Amplicon Sequencing

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**Ion S5 XL Platform for Amplicon Sequencing at Novogene: Advantages and Recommendations**The Ion S5 XL platform utilized by Novogene employs semiconductor sequencing technology, converting chemical signals directly into digital signals on a semiconductor chip. This system operates without laser light sources, optical systems, or cameras. Using unlabeled nucleotides and enzymes for sequencing, it significantly enhances the accuracy of base calling by detecting H+ ions. Compared to the Illumina HiSeq PE250, this platform offers longer read lengths without the need for assembly and shorter seque…

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Common Issues in Microbial Resequencing

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**Differences Between Resequencing and Genome-Based Variation Detection:**In genome-based studies, more variation information can be obtained compared to resequencing, especially in highly variable regions of the genome and for discovering new gene information. Additionally, for strains with distant relationships, the genome-based approach is more ideal due to the presence of numerous highly variable regions. Resequencing, on the other hand, has its main advantage in terms of cost. For evolutionary studies involving a large number of closely related strains, using resequencing results as a fou…

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Common Questions in Metagenomic Sequencing

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**Preparation Considerations for Soil Samples:**During sampling, remove surface debris, use an ethanol-flamed shovel to collect soil from the 5-20 cm underground layer, sieve through a 2 mm mesh after removing visible roots. Combine samples from three collection points, each with 5g of soil, store in sterile centrifuge tubes below 0°C. Upon completion, transport to the lab for DNA extraction. If immediate processing is not possible, freeze the soil samples rapidly at -20°C.**Preparation Considerations for Fecal Samples:**Fecal samples can be stored at -80°C. Internal feces are preferred for…

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Common Issues in Bacterial Genome De Novo Sequencing

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Comparison of Genome and Re-sequencing for Detecting Variations in New Bacterial Strains:Both genome comparison and re-sequencing can be used to detect variations in new bacterial strains, but there are some differences in their applications. Re-sequencing, due to its limited read length, generally retains alignment results with a similarity of 95% or above to ensure accuracy. However, for regions with significant variations, the detection results may be suboptimal. Considering the rapid evolution rate of bacteria and the presence of regions with substantial variation in most strains, genome c…

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Common Questions about ChIP-Seq

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Which species can undergo ChIP-Seq?Species should be diploid, with genes annotated to the chromosomal level (NCBI), and have complete gene annotation (GTF file).What is the relationship between the Input and IP samples?Input and IP are two separate samples, processed in parallel during early detection, library construction, and sequencing. However, in the analysis, the sequencing data from both samples are integrated for comprehensive analysis to obtain the final peak results and subsequent analyses.What is Input, and what is its purpose?Before performing immunoprecipitation, a portion of frag…

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Exome Sequencing Common Issues

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1. What types of research is exome sequencing suitable for?Exome sequencing is primarily suitable for studies focusing on potential variations within the coding regions, known as exons, which constitute about 1-2% of the human genome. With approximately 180,000 exons in the human genome, exome sequencing is particularly effective for investigating diseases caused by variations in coding regions. It offers cost-effective sequencing, especially for high-depth and large-sample studies, identifying both common and rare mutations. It is commonly applied in the research of Mendelian genetic diseases…

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