Illumina Sequencing

GRT Hub houses two Illumina sequencers: NovaSeq6000 and MiSeq. These two sequencers differ in capacity and read length. The NovaSeq6000 has the greater read capacity of the two; generating as much as 10B reads in one run. This makes the cost per read lower than the MiSeq. The MiSeq has a smaller output, however its technology allows it to have a longer read length, which makes it perfect for metagenomics sequencing. Please see below for more information about our sequencers.

NovaSeq6000

The NovaSeq platform offers 4 different flowcell options that differ in available read length, data output, and per read cost: SP (800M reads), S1 (1.6B reads), S2 (4B reads), and S4 (10B reads). Users typically select the S4 flowcell because it is the most economical option, as it can generate up to 10B reads per run, so the cost per read is lower. The smaller flowcells are a good option for users that are willing to offset higher costs for faster turnaround time or for unique libraries since users would be purchasing full flowcells instead of sharing a flowcell with other users.

GRT Hub currently offers two basic options for NovaSeq6000 sequencing on the S4 flowcell: PE100 and PE150. Users that require special read parameters outside the basic options should contact GRT Hub staff to see what options are available. More information about the NovaSeq6000 specification can be found here.

MiSeq

Similar to the NovaSeq, the MiSeq platform offers 4 different flowcell options that differ in available read length, max number of reads, and cost: v2 Nano (1M reads), v2 Micro (4M reads), v2 (12M reads), and v3 (22M reads). Unlike the NovaSeq, each MiSeq run will be for only one project. This allows greater customization for read parameters. The only confusing aspect of the MiSeq is that the read length options are dependent on the flowcell selected. For example, the v2 Nano has a max capacity of 500cycles (not including index), so the largest read parameter would be a PE250. GRT Hub's iLab sequencing request form will auto populate a dropdown of available read lengths based on the flowcell chosen,  however more information about the MiSeq's specifications can be found here.

RNA Library Construction

IMPORTANT: Before submitting your RNA samples, please ensure that all samples have been properly Dnase I treated. Most columns can remove gDNA during extraction, however purified samples still need to undergo an extra treatment and clean up with Dnase I to properly remove as much gDNA from the sample as possible.

GRT Hub offers several library construction options depending on the source and quality of the RNA sample:

  • Poly-A Enrichment Library Construction: Illumina TruSeq Stranded mRNA Kit
    This type of library construction enriches for mRNA by hybridizing the poly A-tail on mRNA with the poly-T oligos on magnetic beads. This method requires high quality total RNA, with a RIN score greater than 8. Input range is 100 - 1000ng of total RNA. Please reference this protocol (link) for more information.
  • rRNA Depletion Library Construction: Illumina TruSeq® Stranded Total RNA Library Prep Gold
    This type of library construction removes ribosomal RNA from the total RNA sample. This method will enrich for non-ribosomal poly-adenylated and non-polyadenylated RNA.  The rRNA depletion workflow can be used with degraded RNA. Input range is 100 - 1000ng of total RNA. Please reference this protocol (link) for more information.
  • Low Input RNA Library Construction
    The previously mentioned Illumina RNA sequencing construction kits are the standard RNA-seq kits used; however, their input range is rather high. For samples with very low RNA concentrations, the GRT Hub does offer low input options. However, please keep in mind that these low input kits work by increasing the amount of PCR amplification, so there may be higher rates of PCR bias. Quantification for these low concentration RNA samples is also difficult due to the lower limits of typical RNA quantification assays, so input amounts can be more variable.

    • The following are the two low input kits available at GRT Hub:

      1. "SMARTer® Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian"
        Makes cDNA from all the total RNA, then depletes the rRNA cDNA. The remaining cDNA are made into libraries. It also contains an additional 8 nt unique molecular identifier (UMI) to account for PCR bias. (ribo depletion, stranded, UMI). Input Range: 250pg - 10ng (FFPE 500pg - 10ng) in a max input volume of 8ul
      2. "SMART-Seq® v4 Ultra® Low Input RNA Kit"
        Makes cDNA from total RNA using the poly A tail. The resulting cDNA is made into libraries using the Nextera XT kit. (poly A, non-stranded). Input Range: 10pg - 10ng (FFPE 500pg - 10ng) in a max input volume of 9.5ul
  • Micro RNA and Small RNA Library Construction: PerkinElmer NEXTflex Small RNA-Seq Kit v3
    GRT Hub also offers Micro RNA and Small RNA library construction from total RNA. However, please ensure that the RNA isolation method retains micro and small RNA. Input range: 1 ng - 2 µg total RNA or purified small RNA from 1-10 µg total RNA in up to 10.5 µL. Please reference this protocol (link) for more information.