Here are our favorite resources about next generation sequencing (NGS) as well as information about the sequencing platforms we house. If you’re new to NGS, please check out this beginners guide.
We measure DNA concentrations with Qubit and band sizes for possibly degraded DNA with the Agilent Bioanalyzer. Here is quick refresher on genomic DNA quantity and quality.
*Please note: NanoDrop Spectrophotometers can overestimate concentrations. Take this into consideration if submitting samples that have been quantified this way.
For Illumina sequencing, we recommend the following kits for genomic DNA extractions:
For long read sequencing, we recommend the following kits for high molecular weight genomic DNA extractions:
We measure RNA concentrations with Qubit and band sizes with the Agilent Bioanalyzer. Here is a quick refresher on RNA quality. We strongly recommend DNase treatment on RNA samples.
*Please note: NanoDrop Spectrophotometers can overestimate concentrations. Take this into consideration if submitting samples that have been quantified this way.
We recommend the following kits for RNA extractions:
Short Read Sequencing
Illumina’s Next Generation Sequencing technology uses sequencing by synthesis (SBS) chemistry and allows for both single-read and paired-end sequencing options up to 300 bp in length.
We offer short read sequencing services on our Illumina NovaSeq 6000, MiSeq, and NextSeq 1000. As all Illumina sequencers use similar chemistry, same library preparations can be used across these instruments, allowing us to tailor to researchers’ sequencing needs according to read length, throughput, and turnaround time. Illumina provides sequencing coverage recommendations to help determine how many reads are needed for an experiment.
Multiple samples can be indexed by a unique nucleotide sequence and pooled into an individual run to save costs. Samples may be indexed using Illumina’s TruSeq indices, Nextera indices, or custom indexing adaptors. The SGT can demultiplex the reads and provide fastq files for each individual sample if Illumina indices are used.
*Please note: If submitting customer prepared libraries, each library must contain a unique index barcode to allow for pooling and accurate demultiplexing. Indexes cannot be reused for libraries in the same pool.
The NovaSeq 6000 is a robust, production-scale sequencer from Illumina that utilizes patterned flow cells and 2-color channel chemistry. The 2-color channel SBS technology only requires two images to determine all four base calls, which reduces cycle and data processing times while still producing the same high quality data. The NovaSeq 6000 also allows the flexibility to sequence by flow cell or lane. S Prime, S1, and S2 flowcells have 2 lanes while the S4 flowcell has 4 lanes.
SEQUENCING OUTPUT PER FLOW CELL
| NovaSeq 6000 System | ||||
|---|---|---|---|---|
| Flow Cell Type | S Prime | S1 | S2 | S4 |
| 2 × 50 bp | 65–80 Gb | 134-167 Gb | 280–333 Gb | NA† |
| 2 × 100 bp | 134–167 Gb | 266-333 Gb | 560–667 Gb | 1600–2000 Gb |
| 2 × 150 bp | 200–250 Gb | 400-500 Gb | 850–1000 Gb | 2400-3000 Gb |
| 2 X 250 bp | 325-400 Gb | NA† | NA† | NA† |
Specifications based on Illumina PhiX control library at supported cluster densities.
† NA: not applicable
CLUSTERS PASSING FILTER PER FLOW CELL
| NovaSeq 6000 System | ||||
|---|---|---|---|---|
| Flow Cell Type | S Prime | S1 | S2 | S4 |
| Clusters* | 650–800 M | 1.3-1.6 B | 3.3-4.1 B | 8-10 B |
* If you are sequencing paired-end, each cluster will generate two reads. If you are sequencing single-end, each cluster will generate one read.
The MiSeq is a benchtop sequencer from Illumina that allows for more focused applications, such as targeted gene sequencing, metagenomics, small genome sequence, targeted gene expression, and amplicon sequencing. The ability to run long paired-end reads (2 x 250bp or 2 x 300bp) makes this instrument ideal for amplicon sequencing. The MiSeq is capable of generating ~12 million reads passing filter for v2 kits (~24 million for paired-end sequencing) and ~22 million reads passing filter for v3 kits (~44 million for paired-end sequencing).
MiSeq Standard Runs *
| MiSeq v2 kits | Run Time | Clusters Passing Filter | Pass Filter Output |
|---|---|---|---|
| Single-end 50 bp | ~1 day | ~12 M/run | ~0.6 Gb/run |
| Paired-end 2 x 25 bp | ~1 day | ~24 M/run | ~0.6 Gb/run |
| Paired-end 2 x 150 bp | ~1.5 days | ~24 M/run | ~3.6 Gb/run |
| Paired-end 2 x 250 bp | ~2 days | ~24 M/run | ~6 Gb/run |
| MiSeq v3 kits | |||
| Single-end 1 x 150 bp | ~1.5 days | ~22 M/run | ~3.3 Gb/run |
| Paired-end 2 x 75 bp | ~1.5 days | ~44 M/run | ~3.3 Gb/run |
| Paired-end 2 x 300 bp | ~3 days | ~44 M/run | ~13.2 Gb/run |
*Specifications based on Illumina PhiX control library. Yield can vary depending on library type. Run times correspond to sequencing only and do not include primary data processing. Performance may vary based on sample quality, cluster density and other experimental factors.
If you need fewer reads for your project or for testing your library, nano and micro kits are available:
MiSeq Nano and Micro Runs *
| Read length | Run Time | Clusters Passing Filter | Pass Filter Output |
|---|---|---|---|
| Paired-end 150 bp Nano | ~1 day | ~2 M/run | ~0.3 Gb/run |
| Paired-end 250 bp Nano | ~1 day | ~2 M/run | ~0.5 Gb/run |
| Paired-end 150 bp Micro | ~1 day | ~6 M/run | ~1.2 Gb/run |
*Specifications based on Illumina PhiX control library. Yield can vary depending on library type. Run times correspond to sequencing only and do not include primary data processing. Performance may vary based on sample quality, cluster density and other experimental factors.
The NextSeq 1000 is a new benchtop sequencer from Illumina that utilizes a patterned flow cell, 2-color channel chemistry, and an integrated sequencing cartridge. The patterned flow cell features a novel, high resolution optical system and higher cluster densities to allow for increased data output with greater resolution. Two flow cell types are offered to help cater to coverage needs: P1 and P2.
| NextSeq 1000 P1 Flowcell | Sequencing Output per Flow Cell | Run Time |
|---|---|---|
| Paired-end 2 x 50 bp | 10 Gb | ~10 hrs |
| Paired-end 2 x 100 bp | 20 Gb | ~19 hrs |
| Paired-end 2 x 150 bp | 30 Gb | ~19 hrs |
| Paired-end 2 x 300 bp | 60 Gb | ~34 hrs |
| NextSeq 1000 P2 Flowcell | Sequencing Output per Flow Cell | Run Time |
|---|---|---|
| Paired-end 2 x 50 bp | 40 Gb | ~13 hrs |
| Paired-end 2 x 100 bp | 80 Gb | ~21 hrs |
| Paired-end 2 x 150 bp | 120 Gb | ~29 hrs |
| Paired-end 2 x 300 bp | 180 Gb | ~44 hrs |
*Sequencing output specifications based on a single flow cell using Illumina PhiX control library at supported cluster densities. Performance may vary based on library type and quality, insert size, loading concentration, and other experimental factors. Run times correspond to sequencing only and do not include primary data processing.
Clusters Passing Filter per Flow Cell
| NextSeq 6000 System | ||
|---|---|---|
| Flow Cell Type | P1 | P2 |
| Single-end Reads | 100M | 400M* |
| Paired-ends Reads | 200M | 800M† |
* 300M single-end reads for 2 x 300bp
† 600M paired-end reads for 2 x 300bp
Long Read Sequencing
Long read sequencing, also known as third generation sequencing, involves reading sequences that are thousands of bases long. The longer read length generates a reasonable amount of overlap between sequences for better sequence assembly. We offer long read sequencing on Oxford Nanopore Technologies’ GridION sequencer.
Oxford Nanopore Technologies developed unique platforms for the sequencing of long contiguous DNA strands. This third generation sequencing technology uses flow cells with trans-membrane protein pores to read strands of DNA by base associated electrical signal. Oxford Nanopore library preparation kits allow for the sequencing of native DNA, eliminating PCR bias from the data while maintaining strand methylation status. The long reads generated by Oxford Nanopore Technologies are important for detecting structural variants throughout the genome.
The core operates an Oxford Nanopore GridION Mk1 which runs 5 flow cells at a time. The instrument can generate up to 10Gb of data per flow cell with read length upwards of 100Kb. Output per flow cell can vary greatly based on the quality of the DNA.
RECOMMENDATIONS
To take advantage of this sequencing technology, we recommend that you use our High Molecular Weight Extraction services.
Contact us for pricing and sample submissions
The Mission Bio Tapestri Platform provides single-cell multi-omics assays. This innovative platform analyzes genotype and phenotype simultaneously from the same single cells, which lets you reveal clonal heterogeneity and target comprehensive biomarkers.
*Please note: Customers will be responsible for purchasing and providing pre-designed or custom panels from Mission Bio. Our Tapestri single cell library prep fee does not include the cost of Mission Bio’s panels.
Contact us to discuss which panel fits your project best and for sample submissions.