Description
Material provided: data
Unit definition:
Library preparation: QIAseq Stranded mRNA Select Kit
650 million reads for 2×75 Paired End Length. (suggested at least 3.5 million reads per sample – 12 samples per run). 1 Unit should be adequate for ~16 RNA samples of Anopheles. This allows testing of two experimental conditions + two control conditions, with four replicates of each condition = total 16 RNA samples. Four replicates per condition are recommended. Please do not submit projects with less than three replicates per condition, which would be inadequate for statistical analysis.
Short Description:
RNA-seq allows the gene-expression profiling and the characterization of coding and non-coding transcripts to test differential expression across study groups.
Total RNA-Seq analyzes both coding and multiple forms of noncoding RNA for a comprehensive view of the transcriptome.
Benefits of RNA sequencing analysis:
- Captures both known and novel features
- Allows researchers to identify biomarkers across the broadest range of transcripts
- Enables a more comprehensive understanding of phenotypes of interest
- Allows profiling of the transcriptome across a wide dynamic range
The experiment and the product
The sequencing analysis service for RNAseq total transcriptome Illumina sequencing with bioinformatic analysis (Ref-SKU: S.1.1.IT.149.40 & S.1.6.IT.149.45) based on QIAseq Stranded mRNA Select Kit chemicals provided by Illumina, includes the following sequencing for each sample:
- mRNA purification enrichment
- RNA fragmentation
- First strand synthesis
- Second strand synthesis, end-repair, and A-addition
- Strand specific ligation
- Clean start library amplification
- Normalization and Pooling of library
- Sequencing 2×75 PE on NextSeq 550
Agilent 2100 bioanalyzer trace for the library validation step
Bioinformatics analysis service includes the following for each sample:
- Quality control and pre-processing of raw data
- Mapping to the genome or to the annotated transcriptome (when a reference sequence is available)
- Transcriptome assembly (if reference genome is not available or is incomplete)
- Differential gene expression analysis
- Data visualization including counts plot, MA-plot and gene clustering (if required)
End-user responsibilities
Biological material to be provided by the end-users and described in the dedicated request form.
RNA input recommendations
Illumina library preparation protocol is followed by the service provider for preparing of the RNA libraries.
The kit protocol is optimized for 100ng-1µg of high-quality input total RNA.
Minimal sample size to be provided: 1µg of total RNA per each sample. Minimal volume: 20 ml/molecular grade water with absorbance ratio values of 1.8 – 2.0.
Recommended RNA Integrity Number (RIN) value: ≥ 8. Please make sure that your RNA is free of contaminants (DNA and proteins).
Please note that using less RNA or RNA with very low mRNA content, can result in low yield, over-representation of the 3′ ends of the RNA molecules, or even failure of the protocol.
– REMARKS:
- If the provided sample doesn’t comply with the above requested specifications for “RNA INPUT RECOMMENDATIONS”, the customer will be contacted for a re-sampling.
- In case of failure of the sequencing run, the service will be repeated.
- To compensate the biological variability within the experiment due to RNA, a biological triplicate for each experimental condition is recommended.
Dispatch of samples
- Safe-lock 1.5 – 2 ml tubes properly capped to be used to avoid accidental evaporation or sample contamination during shipping.
- Samples to be labelled correctly.
- Correct temperature must be ensured during the shipment to avoid sample degradation.
- Complete the enclosed Sample Submission form and return together with the samples to: orders@pologgb.com.
Supplier responsibilities
Storage of biological sample
Polo d’Innovazione Genomica Genetica Biologia will store the analysed samples only for 1 month after delivery. Extended storage time is available for a maximum of 4 months and must be requested in advance of the experiments in the service request application.
Storage of data
Polo d’Innovazione Genomica Genetica Biologia waives any responsibility for the storage and backup of the supplied project and sequencing data after delivery to the end-user. Polo d’Innovazione Genomica Genetica Biologia will store the project data only for 1 month after delivery. After expiration of this period, all data are removed from our servers and archived (unless otherwise agreed with the end-user). Your data might be re-imported at an extra cost within the period of 1 year after delivery of the final data. After 1 year, all project data will be deleted permanently from our systems. Extended data storage is available on request at an extra cost and must be requested at project start.
Delivery of results
The bioinformatics results will be sent according to the Modalities of Transfer with a Bioinformatic Reports PDF Files. The bioinformatic analysis will be executed in 45 days from the generated sequencing data.
Modalities of transfer:
- by “WeTransfer” platform for data of up to 2Gb
- by “MegaSync” platform for data ranging from 2Gb to 15Gb
- by HD for data over 15 Gb
Please note that the cost of HD driver is not included. Where requested, the end-user must provide the HD.
For the Illumina technical manual, please see the “QIAseq Stranded mRNA Library Kit Handbook”.
For more information, please contact us.
For technical assistance, contact Polo GGB Technical Support:
Email: techsupport@pologgb.com
Telephone: 0039 0577 381310