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Platforms Available

MiSeq Platforms

Sequencing MISEQThe HTSF has MiSeq Platform to cover the 20M read/ lane study needs. It is very versatile with aHiseq 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.

The below table which compares the various MiSeq Nano and MiSeq capabilities can be found in the Forms and Guides tab, Illumina as part of Illumina Platform Comparison and Specification Table.

Illumina MiSeq System Comparison and Specifications

MiSeq MiSeq
Nickname for system Nano MISEQ
Platform MiSeq, Nano MiSeq
Flowcells processed 1 1
Depth 300-500 Mb 0.3 – 15 Gb
Run time 5-24 hr 5 – 65 hr
Lanes/flowcell 1 1
Max PE Reads/LANE 2 million 25 million
Max Clusters/LANE 1 million 12.5 million
Read Type Format Single or Paired End Single or Paired End
Read Length Available 50X, 100X, 150X, 250X, 300x 50X, 100X, 150X, 250X, 300x
Guaranteed read #/lane(see note below) V2 chemistry only
  • 15M , single end,   v3 Chemistry
  • 30 M, Paired end,   v3 Chemistry,
  • 8 M, single end,       v2 Chemistry,
  • 16 M , paired end,   v2 Chemistry
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
NextSeq 2000

The NextSeq 2000 is a mid range Illumina sequencing platform. There are multiple flowcell versions which each have different data yields/ lane.The NextSeq works well for:

  • Single Cell Gene Expression
  • Small Scale Whole Genome Sequencing (WGS)
  • Small Scale Whole Exome Sequencing (WES)
  • Shotgun Metagenomics

A laboratory technician works with the NextSeq 2000 systemPlease view the table below to determine the best fit for your needs. If you have issues determining the best fit, HTSF is here to help you figure out the best format for your study’s data need. There are 3 different flowcell sizes (P1, P2, P3). 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 NextSeq 2000:Material is inserted into the NextSeq 2000

  • It uses patterned flowcells and 2 color channel chemistry.
  • This system replaced the HiSeq platforms with some version similar in data output size, but at a lower cost/ run.
  • With a single lane, sample can be loaded as soon as the machine is available.
  • To determine the best version for your study’s needs, a conference with the HTSF is required.

The below table which compares the various NextSeq 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, DropSeq).

 

NextSeq 2000
Platform P1 P2 Reagents P3 Reagents
Nickname for system

NextSeq P1

NextSeq P2

NextSeq P3

Flowcells processed

1

1

1

Lanes/flowcell

1

1

1

Max PAIRED END Reads/Flowcell

200 M

800 million

2.2 billion

Max SINGLE END Reads/Flowcell

100 M

400 M

1.1 B

Clusters/Flowcell

100 M

400 M

1.1 B

Read Type Format Available

Paired End

Paired End

Paired End*

*some single end runs available upon request
Read Length Available

150x, Custom

50x, 100x, 150x, Custom

50x*, 50x, 100x, 150x, Custom

na

na

*1×50 available

Guaranteed Data Yield/ Flowcell

100M Clusters

400M clusters

1.1 B clusters

200M Paired End Reads

800M paired end  Reads

2.2 billion paired end Reads

Key applications

Small WGS, WES, scRNA-Seq

Small WGS, WES, scRNA-Seq

Small WGS, WES, scRNA-Seq, smallRNA/miRNA-Seq

 

NextSeq 2000 Requirements

P2 Reagents

P3 Reagents

Number of Lanes

1

1

Minimum Volume (ul)

15

15

Molarity (nM), preferred

5

5

Molarity (nM), min to make with NO dilution in prep to run FC

3

3

Number of Clusters/Flowcell

400M

1.1B

Number of Single End Reads Per Flowcell

400M

1.1B

 

Recommended Number of Samples per Pool for Each NextSeq Platform
Small whole-genome sequencing (300 cycles) 130 Mb genome; > 30× coverage

30

82

Whole-exome sequencing (200 cycles) 50× mean targeted coverage; 90% targeted coverage at 20×

16

44

Shotgun Metagenomics (300 cycles) 50M reads/sample

8

20

Single-cell RNA-Seq (100 cycles) 4K cells, 50K reads/cell

2

5

miRNA-Seq or small RNA analysis (50 cycles) 11M reads/sample

n/a

96

NovaSeq 6000 Platform

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.

novaseq

  • 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
Lanes/flowcell 2 2 2 4
Max PE Reads/Flowcell 1600 million 3.2 billion 7.6 billion 20 billion
Max Clusters/Flowcell 800 million 1.6 billion 3.8 billion 10 billion
Read Type Format Paired End

*single end available upon request

Paired End
Read Length Available* 50x, 100x (SE), 150x 50x, 100x, and 150x 50x, 100x, and 150x 100x, 150x
Guaranteed read #/Flowcell (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
Flowcells processed 1 1 1 1
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
Lanes/flowcell 2 2 2 4
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,

Sequencing