The Illumina MiSeq is a benchtop sequencer that enables diverse levels of output that range from 1 million to 25 million read-pairs. The instrument supports both single-end (1x 50 cycle) and paired-end (2 x 75 cycle, 2 x 150 cycle, 2 x 250 cycle, 2 x 300 cycle) sequence runs which require one to three days of run time. The MiSeq is an excellent option for small sequencing projects such as 16S metagenomics, HLA sequencing, and targeted custom amplicon sequencing projects.
Sequencing experiments on the MiSeq are ordered on a per flow cell basis. Prior to sequencing, library quality control assays (Invitrogen Qubit Assay, Agilent ScreenTape Assay, qPCR with the Kapa Library Quantification Kit) are performed to qualify libraries and normalize the library pool. Costs for Library QC are included as part of library preparation when the High-Throughput Genomics (HTG) Shared Resource constructs the library. Alternatively, an additional fee is charged per sample or per pre-pooled sample when researchers construct libraries within their lab.
Frequently Asked Questions
- What is the average quantity of sequence reads delivered by a MiSeq flow cell?
- What run types are supported on the MiSeq platform at the HTG Shared Resource?
- What length of index reads are supported on the MiSeq?
- What is the run time on a MiSeq?
- Can I construct my own libraries for sequence analysis on the MiSeq?
- What volume or quantity of a sequencing library is required for sequence analysis on a MiSeq instrument when a researcher constructs their own libraries?
- What recommendations are available for sequencing a low diversity library?
- Should I be concerned if adapter dimer products are present in my sequencing library?
- How do I know if my library contains adapter dimers?
- Can I provide custom primers to be used for sequencing my libraries on the MiSeq?
- What is the optimal insert size for libraries that are sequenced on the MiSeq?
- How is the library’s quality assessed prior to sequence analysis on the MiSeq?
- Can the HTG Shared Resource provide assistance with analysis of sequence data?
- How long will sequencing data from my experiment be available for download on the GNomEx server?
1. What is the average quantity of sequence reads delivered by a MiSeq flow cell?
Read output specifications for MiSeq flow cells are provided below. Please note that low diversity libraries (16S rRNA, amplicon libraries, etc.) are sequenced at a lower cluster density which will reduce the output of sequence reads:
- MiSeq v3 flow cell: 20–25 million read-pairs
- MiSeq v2 flow cell: 10–15 million read-pairs
- MiSeq v2 Micro flow cell: 3–4 million read-pairs
- MiSeq v2 Nano flow cell: 0.7–1.0 million read-pairs
2. What run types are supported on the MiSeq platform at the HTG Shared Resource?
The HTG Shared Resource supports the following run configurations on the MiSeq platform:
- MiSeq 50 Cycle Single-Read Sequencing v2
- MiSeq 150 Cycle Paired-End Sequencing v2
- MiSeq 250 Cycle Paired-End Sequencing v2
- MiSeq 75 Cycle Paired-End Sequencing v3
- MiSeq 300 Cycle Paired-End Sequencing v3
- MiSeq Nano 50 Cycle Paired End Sequencing v2
- MiSeq Nano 150 Cycle Single-Read Sequencing v2
- MiSeq Nano 250 Cycle Paired End Sequencing v2
- MiSeq Micro 150 Cycle Paired End Sequencing v2
4. What is the run time on a MiSeq?
Approximate run times on the MiSeq system are documented below:
- 1 x 50 bp run on an v2 flow cell: 6 hours
- 2 x 150 bp run on an v2 flow cell: 24 hours
- 2 x 250 bp run on an v2 flow cell: 39 hours
- 2 x 75 bp run on an v3 flow cell: 21 hours
- 2 x 300 bp run on an v3 flow cell: 56 hours
5. Can I construct my own libraries for sequence analysis on the MiSeq?
Libraries constructed by individual researchers can be sequenced on the MiSeq. Prior to sequencing, quality control assays will be performed on these libraries including:
- Qubit dsDNA HS Assay
- Agilent ScreenTape Assay
- qPCR with the KAPA Library Quantification Kit
7. What recommendations are available for sequencing a low diversity library?
Examples of low diversity libraries include 16S rRNA libraries, CRISPR libraries and single amplicon libraries. In each of these cases, one or more nucleotides is significantly under-represented during each cycle of the sequence run which can negatively impact accurate base calling within a sequence lane. To overcome the repercussions of low base diversity, a balanced library such as the Illumina PhiX v3 library, can be added to the low diversity library at a molarity that will represent approximately 10–20% of the sequence reads within the lane. The balancer library will then enable sufficient representation of all four bases during each cycle of the sequence run and improve quality scores for the sequence run.
8. Should I be concerned if adapter dimer products are present in my sequencing library?
Adapter dimer products are able to hybridize to a MiSeq flow cell more efficiently than library molecules containing inserts. The researcher should be aware that a disproportionate quantity of adapter-only reads may be present in the sequence data when sequencing such libraries.
10. Can I provide custom primers to be used for sequencing my libraries on the MiSeq?
Custom oligonucleotides can be provided for priming Read 1, Index Read 1 and Index Read 2 on the MiSeq. Although custom primers often result in successful sequence runs, we are unable to guarantee the yield of sequence reads or the success of the run when researchers provide custom sequencing primers.
12. How is the library’s quality assessed prior to sequence analysis on the MiSeq?
Quality control assays are performed to validate libraries prior to sequence analysis on the MiSeq. These assays include the following: Qubit dsDNA High Sensitivity Assay (library concentration), Agilent DNA ScreenTape Assay (size distribution), and qPCR with the Kapa Library Quantification Kit to calculate and normalize the molarity of individual libraries when preparing library pools to load on a MiSeq flow cell.
The cost for these quality control assays is included as part of library preparation when the HTG Shared Resource constructs the library. Alternatively, an additional fee is charged per sample (or pre-pooled sample) when researchers construct libraries within their own lab.
13. Can the HTG Shared Resource provide assistance with analysis of sequence data?
The HTG Shared Resource does not provide sequence analysis services. Please contact the Cancer Bioinformatics Shared Resource for assistance with analysis at bioinformaticshelp@bio.hci.utah.edu.
14. How long will sequencing data from my experiment be available for download on the GNomEx server?
Sequencing data will be available on the GNomEx server for approximately six months. The Cancer Bioinformatics Shared Resource has enabled an option for University of Utah laboratories to migrate sequencing data for long-term cloud storage. Please contact the Cancer Bioinformatics Shared Resource for additional information on long-term storage options. Alternatively, researchers can explore other options for data storage but they should be aware that the GNomEx server is unable to support a solution in excess of six months.
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