Next Generation Sequencing (NGS) has revolutionized genomics, offering a wide range of novel applications at high throughput, genome-wide level. The pMedGR facility is equipped with the Illumina NextSeq 550 system, which combines the power of high-throughput, next generation sequencing with a workflow  that enables rapid sequencing of genomic DNA and transcriptomes in a single run and with the flexibility to switch to low-or high-throughput sequencing as needed.

Services

• Genome Sequencing: gDNA-Seq (de novo, re-sequencing)

• Targeted/Exome sequencing: amplification- or hybridization capture-based enrichment and sequencing of targeted genomic regions or whole exomes, etc.

• Transcriptome Sequencing: mRNA-Seq, ribodepleted RNA-Seq, smallRNA-Seq, etc.

• Chip/FAIRE-Seq, ATAC-Seq, etc.

• Metagenomics (16S rRNA-Seq, shotgun sequencing, etc.)

Benefits of DNA Sequencing With NGS

• Sequences large stretches of DNA in a massively parallel fashion, offering advantages in throughput and scale compared to capillary electrophoresis–based Sanger sequencing.

• Provides high resolution to obtain a base-by-base view of a gene, exome, or genome.

• Delivers quantitative measurements based on signal intensity.

• Detects virtually all types of genomic DNA alterations, including single nucleotide variants, insertions and deletions, copy number changes, and chromosomal aberrations.

• Offers high throughput and flexibility to scale studies and sequence multiple samples simultaneously.​

Benefits of RNA Sequencing With NGS

RNA-Seq with next-generation sequencing (NGS) is the method of choice for researchers studying the transcriptome. It offers numerous advantages over other methodologies:

• Broader dynamic range enables more sensitive, accurate, and cost-effective measurement of gene expression.

• Not limited by prior knowledge—can detect both known and novel features.

• Can be applied to any species, even if reference sequencing is not available.

• Align sequencing reads across splice junctions, and detect isoforms, novel transcripts and gene fusions.

• Identification of diagnostic or prognostic biomarkers based on gene expression signatures under disease states

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Multiplexing
DNA and or RNA samples are tagged with a unique index identifier during sample preparation and a number of samples can be pooled and sequenced together in one lane thus reducing the cost per sample. 

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Libraries provided by users

Please contact Genomics Facility personnel prior to library construction. User-provided starting material and prepared libraries have to meet Genomics Facility QC standards before sequencing.

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Indicative amounts of minimum material required per application

Type of Library ǁ Minimum Amount of Starting Material ǁ Minimum Concentration

3’ mRNA-Seq ǁ >=500 ng ǁ >=50 ng/µl

mRNA-Seq ǁ >=1µg ǁ >=50 ng/µl

Targeted/Exome-Seq ǁ >=500 ng ǁ >=50 ng/µl

Chip/FAIRE-Seq ǁ >=10ng ǁ >=500pg/µl

For user made libraries a final library concentration of min 5ng/ul is required

Quality of Starting material

RNA-Seq: High quality total RNA (BioAnalyzer RIN > 7), 260/280 ~2

Chip-Seq: Purified IP DNA with majority of fragments within size range 200 - 400 bp

FAIRE-Seq: Purified DNA with majority of fragments within size range 200 - 700 bp

gDNA: High quality & high molecular weight DNA

The above amounts and qualities are indicative. Please contact the facility for more details.

Instructions

1.  Planning your assay:  Choose an assay that best addresses your research question.

2.  Collecting/processing your samples:

• The material needed for NGS is nucleic acid, either DNA or RNA. The user can provide DNA or RNA or, for further instructions, can contact our CLINICAL samples reception & processing unit (for collection vessels or extraction protocols)

• Low-quality samples may produce less-than-stellar libraries. In this case, the user is informed and can decide if the sample should proceed to sequencing or not.

3.  Running your samples:  An online order is required before samples can be run. Turnaround time varies depending on the size of the project and current workload.

4.  Data analysis:  check the Bioinformatics analysis pipelines for each assay.