Description
Material provided: data
Unit definition: 10 samples/run
Output for PCR Free Library Prep 4Gb
Output for PCR-Based Library Prep: 8Gb
Short Description: Long-read sequencing technologies can produce long sequencing reads with average fragment lengths of over 10,000 base-pairs and maximum lengths reaching 100,000 base- pairs. Compared with short reads, the assemblies obtained from long-read sequencing platforms have much higher contig continuity and genome completeness as long fragments are able to extend paths into problematic or repetitive regions.
Owing to highly complex of the genomes with many long repetitive elements, copy number alterations, and structural variations, short-read paired-end technologies are insufficient for discovery. Long-read sequencing delivers reads in excess of several kilobases, which can span complex or repetitive regions with a single continuous read, allowing for the resolution of these large structural features. Nanopore utilizes an ionic current through nanopores and measures the changes in current as biological molecules pass through the nanopore or near it. The information about the change in current can be used to identify that molecule. Nanopore sequencing technology to rapid sequence long-read fragments with high quality, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. It combines long read technology and rapid sequencing to produce the highest-quality de novo assemblies, phase alleles and variants, and significantly improve the detection of large structural variants.
Benefits of the Nanopore technologies:
- DIRECT MOLECULAR ANALYSIS: direct sequencing of the native DNA/RNA that helps avoid errors introduced by amplification steps
- SCALABILITY: MinION can be run for minutes or days according to experimental need
- REAL TIME DATA: data starts streaming immediately / stopping sequencing experiments if sufficient data has accumulated to answer a question
The experiment and the product
The service of Long-read DNA sequencing by Nanopore + bioinformatics analysis (Ref-SKU: S.1.5.IT.149.44 & S.1.10.IT.149.48) provides for a comprehensive range of gDNA and amplicon library preparation according to the quantity of starting gDNA.
PCR-Free library preparation
– DNA repair and end-prep
– Adapter ligation and clean-up
– Priming and loading the SpotON flow cell
PCR-based library preparation
– DNA optional fragmentation
– End prep
– Ligation of sequencing adapters and tether attachment
– Loading and sequencing
The bioinformatic analysis of long reads will be achieved using two different approaches based on whether a reference genome is available or not.
Reference-based assembly:
- Quality check and filtering of raw reads;
- Genome assembly by mapping the reads to the reference genome (using the tool minimap2);
- Variant calling, structural and functional annotations (when requested based on the research project);
- Reconstruction of the genome consensus sequence (when requested based on the research project).
De novo assembly:
- Quality check and filtering of raw reads;
- De novo genome assembly and quality control;
- Structural and functional annotation;
End-user responsibilities
Biological material to be provided by the end-users and described in the dedicated request form.
– DNA INPUT RECOMMENDATIONS
- PCR-Free library preparation: The kit protocol is optimized for 1µg of input gDNA total with an absorbance ratio 260/280 of 1.8 – 2.0 and 260/230 of 2.0-2.2 Minimal sample size to be provided 2µg of genomic DNA. Minimal volume: 20 ml/molecular grade water;
- PCR-based library preparation: The kit protocol is optimized for 100ng of input gDNA total with an absorbance ratio 260/280 of 1.8 – 2.0 and 260/230 of 2.0-2.2. Minimal sample size to be provided 200ng of genomic DNA. Minimal volume: 20 ml/molecular grade water;
- Recommended DNA Integrity Number (DIN) value: ≥ 7.
- No detergents or surfactants in the buffer.
– REMARKS:
- If the provided sample doesn’t comply with the above requested specifications for “DNA INPUT RECOMMENDATIONS”, the customer will be contacted for a re-sampling.
- In case of failure of the sequencing run, the service will be repeated.
– DESPATCH 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 sequencing and bioinformatics results will be sent according to the Modalities of Transfer with a QC Library & Bioinformatic Reports PDF Files. The bioinformatic analysis will be executed in 60 working days from the generated sequencing data.
Modalities of transfer:
- by “WeTransfer” platform to email 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 further details on the description of the experiment please consult the publications page of the Nanopore Tech website.
Publications
- “The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community” 2016. Miten Jain, Hugh E. Olsen, Benedict Paten & Mark Akeson. Genome Biology, V17, Article 239.
- “Oxford Nanopore MinION Sequencing and Genome Assembly” 2016. Hengyun Lu, Francesca Giordano, Zemin Ning Genomics, Proteomics & Bioinformatics, V 14, Pages 265-279
For more information, please contact us.
For technical assistance, contact Polo GGB Technical Support:
Email: techsupport@pologgb.com
Telephone: 0039 0577 381310