- Hiseq 2500
- Hiseq 4000
- NovaSeq 6000
MiSeq PlatformsThe HTSF has MiSeq Platform to cover the 20M read/ lane study needs. It is very versatile with a number of different cycle version that can standardly be run (up to paired end, 300 cycles) and can easily be manipulated to run unique or custom cycle formats. In the HTSF hands, MiSeq has been found to be more forgivable about library quality.The ability to run custom cycles and library quality flexibility, make MiSeq especially helpful with R&D library development or pilot assays. It is the workhorse for a variety of applications, including targeted gene, small genome, and amplicon sequencing, ITS and 16S metagenomics. See applications for this platform on the table below.
20M reads too much? HTSF also now offers the MiSeq Nano version for minimal cost. It yields only 1M reads per lane. For example, the HTSF uses the MiSeq Nano as a standard QAQC tool for pools which are planned to be run on the NovaSeq. This small data yield is enough for us to check library and barcode quality of the libraries in the pool. But more critical to a NovaSeq, is the balance of the pool. A Nano run allows us to check the balance, rebalance as needed prior to loading the larger much more expensive platform.
HiSeq PlatformsThe HTSF continues to offer HiSeq platforms. Both the Hiseq2500 and Hiseq4000 have been discontinued by Illumina because they are not considered obsolete. Please view greater information below. The HTSF will continue to offer these platforms until the demand has dropped off or Illumina no longer supports the system, whichever occurs first. We will place announcements on our website and our submissions system, TracSeq, well in advance of the shutdown of these platforms.
The HiSeq 2500 System is a powerful high-throughput sequencing system form Illumina. High-quality data using proven Illumina SBS chemistry. Currently, other than legacy studies, HS2500 allows for 2 lanes to be run in the rapid mode. This is helpful when more data than a MiSeq can supply is required read numbers and unusual cycle format does not study allow a pool to be run on less than 8 lanes with other studies filling the flowcell. We can also run the HS2500 in the v4 high output mode, allowing for 8 lanes of sequencing. This typically only used for legacy studies. We now have more economical methods for you sequence to be run. Please see table below and inquire from the HTSF for best alternatives.
The HiSeq 2500 System is now considered obsolete by Illumina. They intend to continue to provide full support of the instrument and supply the reagents through February 28th, 2023. Because of the HS2500 status with Illumina, reagents have become more expensive. Please inquiry about other options.
The HiSeq4000 System uses a patterned flow cell technology to provide rapid, high-performance sequencing. It has greater data capacity per lane over the HS2500 system and remains at 8 lanes per flowcell. It is the works horse for many studies that do not need the capacity of a NovaSeq SP flowcell or studies which are completing a legacy project. See applications for this platform on the table below.
Illumina will be discontinuing the HS4000 Systems. They will continue to provide full support of the instruments and supply the reagents through March 31, 2024. Because of the HS4000 status with Illumina, reagents have become more expensive. Please inquiry about other options, especially prior to beginning a long-term study.
The below table which compares the various MiSeq and HiSeq abilities can be found in the Forms and Guides tab, Illumina as part of Illumina Platform Comparison and Specification Table.
Illumina MiSeq and HiSeq System Comparison and Specifications
|Nickname for system||Nano||MISEQ||Rapid Run||V4 or High Output||4000|
|Platform||MiSeq, Nano||MiSeq||HiSeq 2500||HiSeq 2500||HiSeq 4000|
|Flowcells processed||1||1||1 or 2||1 or 2||1 or 2|
|Depth||300-500 Mb||0.3 – 15 Gb||50 – 60 Gb (2x100bp)||450 – 500 Gb (2x125bp)||650-750 Gb (2x150bp)|
|Run time||5-24 hr||5 – 65 hr||1 dy – 2 dy||1 dy – 7 dy||1 dy – 3.5 dy|
|Max PE Reads/LANE||2 million||25 million||150 million||250 million||300 million|
|Max Clusters/LANE||1 million||12.5 million||75 million||125 million||150 million|
|Read Type Format||Single or Paired End||Single or Paired End||Single or Paired End||Single or Paired End||Single or Paired End|
|Read Length Available||50X, 100X, 150X, 250X, 300x||50X, 100X, 150X, 250X, 300x||50X, 100X,150x,250x||50X, 100X,125x||50x, 75x, 100x, 150x|
|Guaranteed read #/lane(see note below)||V2 chemistry only||
||100M, single end,
200M, paired end
|130M, single end,
260M, paired end
|200M, single end,
400-450M, paired end
|Key applications||Small genome, amplicon, and targeted gene panel sequencing, confirming complex balanced pools||Small genome, amplicon, and targeted gene panel sequencing, confirming complex balanced pools||Production-scale genome, exome, transcriptome, sequencing, and more||Production-scale genome, exome, transcriptome, sequencing, and more||The same as HiSeq2500 with longer read lengths and more reads|
NovaSeq 6000 Platforms
The NovaSeq 6000 is currently the most powerful Illumina sequencing platform. There are multiple flowcell versions which each have different data yields/ lane. The NovaSeq works well for:
- Large production studies
- Deep sequencing
- Whole Genome Sequencing (WGS)
- Whole Exome Sequencing (WES)
- RNA sequencing ( whole Transcriptome Sequencing)
- Tumor-Normal Profiling
Please view the table below to determine the best fit for your needs. It can be difficult to determine best NovaSeq flowcell version. HTSF is here to help you figure out the best format for your study’s data need. There are 2 pool loading workflows on the NovaSeq. The standard mode has a single pool run over the full flowcell. The XP mode allows different pool to be loaded on each lane. There is an additional fee per lane for the XP mode to be used. There is a more limited number of standard cycle set up. But the HTSF continues to run custom cycles on the NovaSeq.
A few quick view details about the Novaseq6000:
- It uses patterned flowcells and 2 color channel chemistry.
- This system provides users with the speed and flexibility to complete large scale projects faster and with greater output than previous HiSeq systems.
- The NovaSeq provides more data when compared to previous HiSeq systems.
- All NovaSeq version can run a different pool on each lane with the use an XP kit (for an additional fee). Standard loading format has the same pool loaded over all lanes of the flowcell.
- To determine the best version for your study’s needs, a conference with the HTSF is required.
- Due to high cost of NovaSeq flowcells, HTSF requires extra QAQC for all pools to support a successful run.
- QAQC can include qPCR for library concentration determinant.
- QAQC usually includes a MiSeq Nano run to check library and barcodes quality and pool balance. We won’t require a Nano if using SP/PE/50x flowcell, unless it is an extremely large pools of >50 samples.
- NovaSeq requires more material to be loaded that is typically required on HiSeq Platforms. Please discuss with the HTSF what is required if you plan to submit study made libraries or pools.
The below table which compares the various NovaSeq loading version abilities can be found in the Forms and Guides tab, Illumina as part of Illumina Platform Comparison and Specification Table.
Please note, cycles can be made custom to certain library prep methods (i.e. 10x genomics).
NovaSeq Standard Loading (one pool/flowcell)
|Nickname for system||SP||S1||S2||S4|
|Platform||S Prime||NovaSeq 6000 S1||NovaSeq 6000 S2||NovaSeq 6000 S4|
|Flowcells processed||1 or 2||1 or 2||1 or 2||1 or 2|
|Depth||200-250 Gb (2x150bp)||400-500 Gb (2x150bp)||1000-1250 Gb (2x150bp)||2400-3000 Gb (2x150bp)|
|Run time||1 dy – 2 dy||1 dy – 2 dy||1 dy – 3 dy||1 dy –3 dy|
|Max PE Reads/LANE||1600 million||3.2 billion||7.6 billion||20 billion|
|Max Clusters/LANE||800 million||1.6 billion||3.8 billion||10 billion|
|Read Type Format||Paired End
*single end available upon request
|Read Length Available*||50x, 100x (SE), 150x||50x, 100x, and 150x||50x, 100x, and 150x||100x, 150x|
|Guaranteed read #/lane (see note below)||1.4 billion, paired end||3 billion, paired end||7 billion, paired end||17 billion, paired end.|
|Key applications||WGS model organisms, FAIRE/ChIP-seq large pools, metagenomics||Single Trio Human, 10X single cell, Chip-seq transcriptome||Production-scale genome, exome, transcriptome, sequencing, and more||Large production-scale genome, exome, transcriptome, sequencing, and more|
NovaSeq-XP Mode Loading (one pool/lane)
|Nickname for system||SP-XP||S1- XP||S2-XP||S4-XP|
|Platform||NovaSeq 6000 SP XP||NovaSeq 6000 S1 XP||NovaSeq 6000 S2 XP||NovaSeq 6000 S4 XP|
|Depth||125 Gb (2x150bp)||200-250 Gb (2x150bp)||500-625 (2x150bp)||600-750 Gb (2x150bp)|
|Run time||1 dy – 2 dy||1 dy- 2 dy||1 dy – 2 dy||1 dy – 2 dy|
|Max PE Reads/LANE||800 million||1600 million||3.8 billion||5 billion|
|Max Clusters/LANE||400 milion||800 million||1.9 billion||2.5 billion|
|Read Type Format||Paired End. Single End for some cycles||Paired End. Single End for some cycles||Paired End. Single End for some cycles||Paired End|
|Read Length Available||50x, 150x,||50x, 100x, 150x||50x, 100x, and 150x||100x, 150x|
|Guaranteed read #/lane(see note below)||600 million, paired end
|1.4 billion, paired end
|3.2 billion, paired end||4 billion, paired end|
|Key applications||10X single cell, Chip-seq transcriptome||Single Trio Human, 10X single cell, Chip-seq transcriptome||genome, exome, transcriptome, ChIP-seq||genome, exome, transcriptome,|