Client Area > FAQs

NGS


Which sequencing platforms are available at Genoinseq?

Genoinseq provides high-throughput sequencing services with the Ion ProtonTM (Ion Torrent, Thermo Fisher Scientific), and Miseq® and Nextseq®(Illumina®) platforms.


Where can I obtain information regarding the Ion Proton and Miseq/NextSeq Sequencing Systems?

Additional information on these technologies and equipment may be obtained through the company websites, www.thermofisher.com and www.illumina.com


What types of DNA sequencing are available at Genoinseq?

Genoinseq services provide de novo genome sequencing and genome resequencing, exome sequencing, sequencing for microsatellite discovery, amplicon sequencing, plasmid, fosmid and virus de novo sequencing, microbial community profiling and metagenome sequencing. 


What are the differences between the various types of DNA sequencing?

De novo genome sequencing enables access to the genetic information encoded in DNA while genome resequencing identifies genetic differences between individuals. Exome sequencing characterizes the genetic alterations in the coding regions of the genome. This data can be used to search for mutations associated with diseases.

Microsatellite discovery is achieved by the sequencing of a random fraction of the organisms’ genome and further identification of potential microsatellites by specific bioinformatics tools

Amplicon sequencing aims the detection of mutations in known genes. As an example, this approach enables the identification of mutations in genes associated with diseases.

In microbial community profiling, the taxonomic identification of organisms is performed by the sequencing of a specific gene, commonly 16S rRNA gene for bacteria and Internal Transcribed Spacer (ITS) for fungi. In this approach, the diversity and composition of the community are defined by the identification of the cultivable and non-cultivable microorganisms present in the environmental sample. Biodiversity studies can also be applied to eukaryotic communities. In this case, 18S rRNA and cytochrome oxidase I (COI) genes can be used.

Metagenome sequencing explores all genomes present in a particular environmental sample. This approach enables the genome characterization of cultivable and non-cultivable organisms existing in that community.

De novo genome sequencing and resequencing, microsatellite discovery and metagenomics have different objectives but use the same methodology workflow. The sequencing library is prepared by cleaving genomic DNA into small fragments and ligating sequencing adapters onto the ends of each fragment. On the other hand, biodiversity studies and amplicon sequencing require the amplification of a gene or a pool of genes for each sample.


Which platforms are used for DNA sequencing?

Genoinseq provides high-throughput sequencing services with the Ion ProtonTM (Ion Torrent, Thermo Fisher Scientific), and Miseq® and Nextseq®(Illumina®) platforms. After a detailed project analysis, the platform is chosen to best attend the needs of each project and client requirements.


Does Genoinseq perform DNA extraction?

Genoinseq provides this service and uses optimized protocols for DNA extraction from different biological samples. High-quality DNA is essential to obtain the best sequencing results.


What DNA quantification and quality assessment methods are suggested?

DNA quantification should be performed by fluorometry (e.g. Qubit dsDNA HS Assay Kit). Spectrophotometric methods (e.g. Nanodrop) are inaccurate for quantification and not suited for this sequencing approach. The quantification can be biased by the presence of single-stranded DNA, RNA, and other contaminant molecules. On the other hand, the determination of OD260/280 and OD260/230 ratios is recommended for sample purity assessment (e.g. Nanodrop). The DNA integrity should be checked by electrophoresis (e.g. agarose gel).


What if the DNA sample does not fulfill the minimum quality and quantity requirements?

In this case, we suggest you contact Genoinseq so that together we might find the best solution for your project.


What platforms are used for RNA Sequencing?

Genoinseq RNA Sequencing services are provided using the Miseq® and Nextseq®(Illumina®) platforms. Genoinseq may suggest upon request and following project analysis, the platform that best attends the needs of each project and client requirements.


What types of RNA sequencing are available at Genoinseq?

Genoinseq provides several RNA-Seq services which include Whole Transcriptome, mRNA as well as small and micro RNA sequencing. 


What are the differences between the various types of RNA Sequencing?

Depending on the RNA sequencing service requested, distinct RNA enrichment methods are applied.

Total RNA sequencing enables the characterization of all RNA transcripts, coding and non-coding, from an organism or community. As ribosomal RNA represents the majority of the transcriptome, rRNA depletion is necessary before library preparation.

mRNA sequencing aims the characterization of all transcripts based on the enrichment of poly-A tails, characteristic of this type of RNA. For prokaryotes, only rRNA depletion can be applied since their transcripts do not contain poly-A tails.

Small (<200nt) and micro (10-40 nt) RNA sequencing include the non-coding small transcript portion of the transcriptome.


Is it possible to characterize small and microRNAs with Whole Transcriptome sequencing?

Due to the size of small and micro RNAs, these cannot be included in Whole Transcriptome sequencing. If you wish to sequence this type of RNAs we suggest you perform an RNA extraction that enriches for these molecules and choose the small RNA sequencing service.


Does the RNA library preparation maintain the transcript strand orientation (strand-specific)?

The RNA library preparation protocol is strand-specific. By this method, it is possible to accurately characterize and quantify overlapping transcripts that are transcribed on different strands which would not be possible with a non-strand-specific protocol. Stranded information identifies from which of the two DNA strands a given RNA transcript was derived thus providing increased confidence in transcript annotation and enabling the detection of antisense transcript expression.


How should I extract RNA?

The RNA extraction method adopted should purify all RNA species including small RNAs. High-quality RNA samples are essential to obtain good sequencing results. It is crucial you adopt the appropriate standard procedures for RNA handling. The purified RNA should be preferentially eluted or dissolved in nuclease-free water.

If you wish to sequence small/micro RNAs we recommend an extraction kit that selects and enriches these molecules.


I am not able to obtain the minimum quantity of RNA required. How should I proceed?

Please contact our services. Genoinseq can help you find the best solution or suggest alternative protocols for library preparation from limited quantities of RNA.


What procedures does Genoinseq perform to check the quality of the RNA sent?

All samples are submitted to a quality control procedure before library preparation. The sample purity is verified by measuring the OD260/280 and OD260/230 ratios (e.g. Nanodrop), RIN (RNA Integrity Number) is determined by capillary electrophoresis (e.g. Bioanalyzer), and RNA is quantified by a fluorometric method (e.g. Qubit RNA HS Assay Kit).


Why should RNAs have a RIN > 8?

The higher the RIN value, the better the sequencing results. Low RIN values indicate RNA degradation, mainly ribosomal RNA. These rRNA fragments will be sequenced together with the mRNA resulting in a lower sequencing depth of relevant transcripts thus decreasing the quality of the sequencing results. Genoinseq recommends a RIN above 8 for any of the RNA Sequencing approaches.


Why should I treat the RNA sample with DNase?

The contaminant DNA will be sequenced as part of the RNA library prepared. Make sure to use an RNA isolation method that includes DNase treatment to remove contaminant DNA.


What are the procedures done to the RNA samples at Genoinseq?

Upon reception, the sample information submitted and sent are verified, and the RNA immediately stored at -80ºC. You will receive an email confirming the sample admittance.


Does Genoinseq provide Genotyping Services?

Genoinseq provides genotyping services of genes of human and non-human sources. The service may include the study of SNPs of a single or multiple genes. In the case of a large number of genes, genotyping is performed by high-throughput sequencing.


How is the data analysis performed?

Bioinformatics is part of the Genoinseq sequencing platform. We have developed processing pipelines for sequencing data analysis. These pipelines are optimized to suit each project with the most appropriate analysis. One of our objectives is to develop and provide user-friendly formats for final sequencing results access and analysis.


How can I access the sequencing results?

All relevant information regarding the project such as objective, strategy, methodology, tasks, and results will be organized into a final report sent in digital format. The sequencing results are organized in files and also provided in the Genoinseq Cloud. 


Where should I send my samples?

Please send your samples and a Printed copy of the Project Submission Form to:

UC-Biotech
Genoinseq
Parque Tecnológico de Cantanhede, Núcleo 04, Lote 8
3060-197 Cantanhede
Portugal

Can I get a confidentiality agreement (CDA/NDA)?

Yes, we have templates ready for signature, or we can sign your company/institute agreement, after review.
Confidentiality is guaranteed as part of our quotation and general terms and conditions.


Do I own intellectual property of my results?

Yes, we are a service provider and we do not claim any intellectual property.


I cannot find the answer to my question.

If you were not able to find an answer to your question, please contact Genoinseq by email: genoinseq(at)biocant.pt or by phone: (+) 351 231 249 170.


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