Description
Material provided: data
Unit definition: Whole Genome Sequencing
Library Preparation: Illumina DNA Prep or TruSeq Nano Sample Prep Kit
400 million reads for 2×150 Paired End Length equivalent to ca 30X sequence depth for 1 sample of Aedes spp. (estimated genome size: 1.38 Gb) or to ca 100X sequence depth for 1 samples of Anopheles spp. (estimated genome size: 280 Mb)
Short Description: The assembly and the single nucleotide variations analysis are performed using the entire genome or specific pieces by amplicons.
Whole genome sequencing allows the analysis of the entire genome.
Whole-genome sequencing is the most comprehensive method for analyzing the genome. It is a process that determines the complete DNA sequence of an organism’s genome by comparing sequences with a known reference genome. Whole genome sequencing (or resequencing) plays a role in identifying DNA biomarkers such as single nucleotide polymorphism (SNPs), insertions and deletions (Indels), structure variations (SV), copy number variations (CNV) and other genetic changes of the sequenced species with high accuracy, it also provides an unprecedented opportunity for characterizing the polymorphic variants in a population, which comprehensively unravels the underlying mechanisms of species origin, development, growth, and evolution.
Advantages:
- Provides a high-resolution, base-by-base view of the genome
- Captures both large and small variants that might otherwise be missed
- Identifies potential causative variants for further follow-on studies of gene expression and regulation mechanisms
- Delivers large volumes of data in a short amount of time to support assembly of novel genomes
Applications:
- Identification of functional genes, biomarkers
- Detection of genome-wide genetic variants
- Associate DNA variants with a trait
- Comparative analysis at the genomic level
The Experiment and the Product
This product includes both Whole Genome DNA Illumina sequencing sequencing (Ref-SKU: S.1.2W.IT.149.41) and bioinformatic analysis service (Ref-SKU: S.1.7W.IT.149.45).
Whole Genome Sequencing, using the Illumina DNA Prep or TruSeq Nano Sample Preparation chemicals including the following for each sample:
- Tagmentation of genomic DNA to convert into adapter-tagged libraries
- Clean-Up of Tagmentated DNA
- PCR Amplification to add index sequences on both ends of the DNA
- Clean-Up of DNA libraries
- Normalization and library pooling
- Sequencing: 2×150 PE on NextSeq 550
The bioinformatic analysis of Illumina reads will be achieved using two different approaches based on whether a reference genome is available or not.
Reference-based assembly:
- QC and pre-processing procedures including trimming of low-quality bases and technical sequences;
- Alignment of reads to a reference genome
- 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);
- Population genomics analysis (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 Illumina library preparation protocol is followed by the service provider for preparing genomic DNA libraries.
Whole Genome Sequencing:
The kit protocol is optimized for 100 ng of input DNA total with an absorbance ratio values of 1.8 – 2.0.
Minimal sample size to be provided 200ng of genomic DNA. Minimal volume: 10 ml/molecular grade water.
Recommended DNA Integrity Number (DIN) value: ≥ 7.
“Genomic DNA”
Material to provide:
- 100ng of gDNA. Minimal volume of 10 ml/molecular grade water. Recommended DNA Integrity Number (DIN) value: ≥ 7;
- Primer sequences.
– 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 “Illumina DNA Library Prep Reference Guide” or the “TruSeq Nano Reference Guide“.
For more information, please contact us.
For technical assistance, contact Polo GGB Technical Support:
Email: techsupport@pologgb.com
Telephone: 0039 0577 381310