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 small genome sequencing, 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, Kapa BioSystems qPCR) are performed to qualify individual libraries and normalize the library pool. Costs for this service are included as part of library preparation when the 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 volume of sequence reads delivered by the MiSeq 2500 flow cell?
Read output specifications for MiSeq flow cells are provided below. Please note that low diversity libraries are sequenced at a lower cluster density which will reduce the output of sequence reads:
- MiSeq v3 flow cell: 20 to 25 million read-pairs
- MiSeq v2 flow cell: 12-15 million read-pairs
- MiSeq v2 Micro flow cell: 3-4 million read-pairs
- MiSeq v2 Nano flow cell: 0.7 to 1.0 million read-pairs
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 Nano 150 Cycle Paired End Sequencing v2
- MiSeq Nano 250 Cycle Paired End Sequencing v2
- MiSeq Micro 150 Cycle Paired End Sequencing v2
- MiSeq 75 Cycle Paired-End Sequencing v3
- MiSeq 300 Cycle Paired-End Sequencing v3
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.
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 Bioinformatics Shared Resource (email@example.com) for assistance.
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, these libraries will experience multiple quality control assays which include a Qubit dsDNA HS Assay, Agilent ScreenTape Assay, and Kapa BioSystems qPCR. Although we highly qualify all libraries that are sequenced on the Illumina platform, we are unable to guarantee the yield of sequence reads when individual researchers construct their own sequencing libraries due to variability in library quality that is outside of our control.
What volume or quantity of a sequencing library is required for sequence analysis on a MiSeq instrument when a researcher constructs their own libraries?
We recommend providing a volume of 10-15 mL for each sequence library (or pool of libraries) at a minimum molarity of 3 nM.
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 volume of adapter-only reads may be present in sequence data delivered when sequencing these libraries.
How do I know if my library contains adapter dimers?
Adapter dimers exhibit a size distribution of approximately 140 to 160 bp in libraries that include dual indexed adapters. Adapter dimers in single indexed libraries are approximately 120 to 130 bp.
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.
What is the optimal insert size for libraries that are sequenced on the MiSeq?
MiSeq flow cells can efficiently support insert sizes that range from 20 bp to 800 bp in size. As inserts extend beyond 1000 bp, the library becomes less effective at hybridizing and efficiently amplifying on the flow cell.
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 ScreenTape Assay (size distribution), and Kapa BioSystems qPCR to calculate molarities and normalize libraries prior to pooling. Costs for these quality control assays are included as part of library preparation when the HTG Shared Resource constructs the library. Alternatively, an additional fee is charged per sample when researchers construct libraries within their lab.
What length of index reads are supported on the MiSeq?
Standard index read lengths on the MiSeq include 1 x 8 bp, 2 x 8 bp and 2 x 10 bp. However, the MiSeq is able to support customized configurations of index read lengths.
What recommendations are available for sequencing a low diversity library?
Examples of low diversity libraries include bisulfite-treated DNA libraries, 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 impact of low diversity, a balanced library such as the Illumina PhiX v3 library, can be added to the lane at a molarity such that it will represent approximately 15-20% of the sequence reads within the lane.