Skip to Main Content

Illumina - NextSeq500 Next Generation

Illumina NextSeq500 Sequencing

Illumina Inc. is arguably the global leader in next-generation sequencing solutions in breadth of applications, in quality of data and in reliability of instrumentation. At the UMass Lowell Biomolecular Characterization Laboratory we have a dedicated sample preparation and NGS expert to help you achieve your project goals on the NextSeq500. We are happy to offer free up-front consultation about clients’ planned experiments. Below is a simplified workflow outlining the process of completing an NGS project at the Biomolecular Characterization Laboratory.

  1. Consult the core about proposed experiment and complete experiment design.
  2. Extract and QC nucleic acid template (RNA or DNA) for Illumina library preparation chemistry.
  3. Convert your nucleic acid material into Illumina libraries at the core.
  4. QC, normalize, and load your libraries into the NextSeq500 and sequence.
  5. Data is delivered as demultiplexed FASTQ files to the Illumina BaseSpace cloud, or by FTP to the UMass Lowell server.

The Illumina NextSeq500 is capable of producing single-end sequences or more commonly, paired-end sequences of various lengths up to 300 bp single-end, or 150 bp paired-end.

Currently, the turn-around-time for samples is about 7 days from nucleic acid delivery through raw data generation.

The Biomolecular Characterization Laboratory is unique in that we provide three modes of usage. Clients can elect to be trained by the expert staff on sample QC, library preparation, and sequencer setup and operation (samples provided by client). Once a client is sufficiently trained (generally 2-3 training sessions, the client will qualify for a reduced self-use rate.) Alternatively, the client can elect to use the core for “service” and have the expert staff complete sample QC through sequencing for the client.

Financial and project consultation is free. If you would like to discuss your project with core staff and/or with Illumina, or if you would like a cost estimate, you can setup an appointment by emailing: Jack_Lepine@uml.edu.

If you would like a letter of support from the Biomolecular Characterization Laboratory, please contact the director of UMass Lowell Core Research Facilities, Teri Hamelin, at: Theresa_Hamelin@uml.edu.

More Information

Sample Requirements for NextGen Sequencing

DNA or RNA should be isolated with any number of commercial isolation kits that are readily available, or by Trizol to ensure extraction of high quality and high quantity DNA or RNA.

The core strongly encourages that the DNA or RNA is quantitated at the core by fluorometry such as on the core’s Life Technologies Qubit 3.0. This ensures the specific quantitation of “usable” DNA.

DNA or RNA should be eluted in or resuspended in 1X TE buffer or in nuclease-free water.

The integrity of the extracted DNA or RNA will be assessed using the Agilent Bioanalyzer at the core. RNA with an RNA Integrity Value (RIN) of 8.0 or higher will proceed to library preparation. Any sample with a RIN below 8.0 will NOT be moved on to library preparation unless specifically requested by the PI.

All initial quantitation, and integrity data will be saved as an Excel spreadsheet and samples will be tracked through the completion of the sequencing run and provided as a report with the de-multiplexed FASTQ files from the sequencing run.

Illumina TruSeq Stranded mRNA Library Prep (Cat# RS-122-2101)

If you are studying coding RNA to determine +/- expression of genes, relative expression levels of genes, splice variants, or novel transcripts, this is the kit for you. Samples can be single indexed or dual indexed (Cat# RS-122-2103) depending on how many samples will be multiplexed in a single run.

To find out more, please visit Illumina's TruSeq Stranded mRNA Library Prep Kit webpage.

Sample requirements:

  • 100 ng – 4 ug of total RNA in <50 ul of nuclease-free water or 1X TE buffer.
  • 10 ng – 400 ng of rRNA depleted or Poly(A)-enriched RNA

Samples that fail initial quant. and qual. will only be converted to libraries at the discretion of the project PI.

Illumina TruSeq Nano DNA Library Prep (Cat# FC-121-4001)

If your study is focused on detecting genomic variants, chromosome rearrangements, Insertions/Deletions (INDELS) or copy number variants (CNV) this is the kit for you. This kit is excellent for low amounts of input DNA, yielding excellent results with as low as 100 ng of input DNA.

To find out more, please visit Illumina's TruSeq Nano DNA Library Prep Kit webpage.

Sample requirements:

  • 100 ng – 200 ng of total RNA in < 52.5 ul of nuclease-free water or resuspension buffer

Samples that fail initial quant. and qual. will only be converted to libraries at the discretion of the project PI.

Illumina TruSeq Small RNA Library Prep (Cat# RS-200-0012)

If you are trying to discover new smallRNAs or micro RNA (miRNA), this is the kit for you. You can enrich smallRNA species (18nt – 30nt in length) from total RNA by targeting the unique 5’ phosphate and 3’ hydroxyl group on small RNA species. This includes most mature miRNA, siRNA, piRNA, and some pre-miRNA. The enriched smallRNA will be converted to cDNA and then converted into libraries for NGS.

To find out more, please visit Illumina's TruSeq Small RNA Library Preparation Kits webpage.

Sample requirements:

  • 1 ug – 20 ug of total RNA in 5ul of nuclease-free water.
  • 10 ng – 50 ng of purified small RNA

Samples that fail initial quant. and qual. will only be converted to libraries at the discretion of the project PI.

Illumina TruSeq Stranded Total RNA Library Prep (Cat# RS-122-2201)

(RiboZero: H/M/R)

If you would like to sequence total RNA (coding and non-coding) from human, mouse, or rat samples excluding rRNA, this is the kit for you. Ideal for detecting gene-fusions, novel transcripts, singlenucleotide polymorphisms (SNPs), and transcript variants. Multiple versions of RiboZero exist that are tailored to various applications, please inquire if standard RiboZero is not the best for your project.

To find out more, please visit Illumina's TruSeq Stranded Total RNA Library Prep Kit webpage.

Sample requirements:

  • 0.1 – 1 ug high-quality total RNA in nuclease-free water

Samples that fail initial quant. and qual. will only be converted to libraries at the discretion of the project PI.