GENOMICS & 

TRANSCRIPTOMICS

UNIT

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Next Generation Sequencing (NGS)

Illumina NextSeq 550

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

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. 

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.

Indicative amounts of starting material required per application

Type of Library ǁ Amount of Starting Material ǁ Concentration

3’ mRNA-Seq ǁ up to 500 ng ǁ as low as 100 pg/µl

mRNA-Seq ǁ min 2µg ǁ >500ng/µl

Chip/FAIRE-Seq ǁ min 10ng ǁ 500pg/µl

Target-Seq ǁ min 3µg ǁ >50ng/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.

 

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