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Sequencing

NovaSeq 6000

The NovaSeq 6000 is the latest advancement in Illumina's line of NGS sequencing instrumentation which combines high throughput patterned flow cell technology with 3 flow cell configurations while drastically reducing run times, simplifying workflow and reducing hands-on time. The NovaSeq allows the Genome Sequencing Facility (GSF) to provide increased throughput and flexibility by operating with multiple flow cell configurations. The Paired End sequencing on the NovaSeq 6000 will be available using the following flow cell configurations: S1 (0.5 Tb), S2 (1.0 Tb), S4 (3.0 Tb) - yielding 0.5 Tb - 6 Tb of sequence data per run on the dual flow cell capable sequencer. The following is a comparison of expected output and data quality for the NovaSeq 6000 flow cell options.

Flow Cell Type S1 S2* S4
Reads Passing Filter – Single FC 1.6 B 2.8 – 3.3 B 10 B
Data Output      
2 x 100 bp 333 Gb 560–667 Gb Not Supported
2 x 150 bp 500 Gb 850 – 1000 Gb 3000 Gb
Performance (Q30)      
2 x 100 bp N/A ≥ 80% Not Supported
2 x 150 bp N/A ≥ 75% N/A
Run Time
2 x 100 bp N/A ~ 25 hr. Not Supported
2 x 150 bp N/A ~ 36 hr. ~ 44 hr.

*The NovaSeq reagent kit supporting the S1 has not been released, therefore performance metrics are subject to change. Anticipated release of S1 is Q1 2018.

What is Index Hopping?

Index hopping (index switching / index drift) has impacted NGS technology since the introduction of sample multiplexing. It can lead to sample misassignment of libraries from the expected index to a different index in a multiplexed pool. Slightly elevated hopping can be experienced on instruments using patterned flow cells like the NovaSeq 6000 which employ exclusion amplification chemistry (EXamp). Index hopping on a patterned flow cell system can range from 0.1 - 2% depending on the type, quality and preparation of the library. Libraries containing higher levels of unincorporated adaptors increase the risk of higher levels of index hopping. The following Best Practices will be implemented by the Genomics Core to minimize index hopping. Investigators preparing their own libraries must follow the Best Practices.

Best Practices

  • Generate libraries using unique dual indexing (UDI). Both the i5 and i7 indexes must be unique within the individual library. The i5 and i7 indexes must also be unique from indexes used in other libraries.
  • Remove free adaptors from library preps: Add 2nd post PCR amplification bead clean-up step or spin column clean-up.
  • Recommended strategies for unique dual index designs (PDF).
  • Store libraries individually at -20°C.
  • Pool libraries prior to sequencing.

For additional information please visit the following web links:

HiSeq 2500

The Illumina HiSeq 2500 sequencing services provided by the Genome Sequencing Facility (GSF) allows for both the standard High Output 8 lane flow cell run and the 2 lane flow cell Rapid Run. Both services are supported by Paired-End (PE) or Single Read (SR) sequencing using the Illumina TruSeq SBS - HS and TruSeq Rapid SBS - HS chemistry. The High Output runs are available indexed or non-indexed as 50 or 100 cycle PE or SR. The Rapid Run is available indexed or non-indexed as 50, 100 or 150 cycle PE or SR. Flow cell clustering is performed using the cBOT cluster generation station. The following information is a comparison of expected output and data quality for both High Output and Rapid Read runs.

Rapid (2 lane)High Output (8 lane)
Cluster Density 750-900 k/mm2 750-850 k/mm2
Clusters/Lane (PF) 130-150M 187-210M
Flow Cell Output (2x50bp) 25 - 30Gb 150Gb
Flow Cell Output (2x100bp) 50-60Gb 300Gb
Flow Cell Output (2x150bp) 75-90Gb Not Supported
Bases >Q30 (2x50bp) 85% 85%
Bases >Q30 (2x100bp) 80% 80%
Bases >Q30 (2x150bp) 75% Not Supported

Run times between the Rapid Read and High Output are significantly different. The following provides a comparison of run times.

Run FormatRapid ReadHigh Output
50 ~ 8 hours ~ 2 days
2 x 50 ~ 16 hours ~ 5 days
2 x 100 ~ 27 hours ~ 10 days
2 x 150 ~ 40 hours Not Supported

Library Preparation

Sequencing library construction is available through the GSF using the Illumina TruSeq Sample preparation kits. The following library services are offered:

  • TruSeq Stranded mRNA - Generate mRNA libraries directly from total RNA that provide strand origin for sequenced mRNA transcripts. Oligo dT capture based method.
  • TruSeq Stranded Total RNA – Generate mRNA and non-coding RNA libraries from total RNA that provide strand of origin for sequenced transcripts. Ribosomal reduction based method.
  • TruSeq RNA Access – Convert total RNA into template molecules of know strand of origin coupled with sequence specific probe capture of coding mRNA. Allows interrogation of low yield / low quality RNA.
  • TruSeq DNA Nano - Generate Libraries from Genomic DNA of limited quantity.
  • TruSeq DNA PCR-Free – Generate libraries without PCR amplification reducing potential bias or gaps.
  • TruSeq ChIP-Seq DNA - Selectively sequence DNA sequences bound by target proteins.
  • TruSeq Small RNA - Generate small RNA / miRNA libraries directly from total RNA.
  • TruSeq Rapid Exome - Ideal for scalable exome sequencing studies.
  • TruSeq DNA Methylation – Converts post bisulfite-treated ssDNA into an Illumina Sequencing library for interrogation of methylation patterns of all CpG, CHH and CHG regions.
  • Reduced Representation DNA Methylation – endonuclease restriction enrichment of methylation rich regions to reduce sequence coverage demand. 

For other library constructs please inquire.

Last modified: Jul 26, 2018
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