Illumina

The 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.

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.

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

Please 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:

• 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

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
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,