Sequencing Overview
NovaSeq 6000 + v1.5 SBS Chemistry
The NovaSeq 6000 is the latest advancement in Illumina's line of NGS sequencing instrumentation which combines the v1.5 SBS chemistry with the high throughput patterned flow cell technology which drastically reduces run times, simplifies workflow and reduces hands-on time. The NovaSeq allows the Genome Sequencing Facility (GSF) to provide increased throughput and flexibility by operating with multiple flow cell configurations and XP Chemistry capability. XP Chemistry allows independent lane addressment of the NovaSeq flow cells. The Paired End sequencing on the NovaSeq 6000 will be available using the following flow cell configurations: SP (0.4 Tb), S1 (0.5 Tb), S2 (1.25 Tb), S4 (3.0 Tb) - yielding 0.8 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 | SP | S1 | S2 | S4 |
|---|---|---|---|---|
| PE Reads Passing Filter per FC |
1.3 - 1.6 B | 2.6 - 3.2 B | 6.6 - 8.2 B | 16 - 20 B |
| Data Output | ||||
| 2 x 50 bp | 65 - 80 Gb | 134 - 167 Gb | 333 - 417 Gb | N/A |
| 2 x 100 bp | 134 - 167 Gb | 266 - 333 Gb | 667 - 833 Gb | 1600 - 2000 Gb |
| 2 x 150 bp | 200 - 250 Gb | 400 - 500 Gb | 1000 - 1250 Gb | 2400 - 3000 Gb |
| 2 x 250 bp | 325 - 400 Gb | N/A | N/A | N/A |
| Performance (Q30) | ||||
| 2 x 50 bp | ≥ 85% | ≥ 85% | ≥ 85% | N/A |
| 2 x 100 bp | ≥ 80% | ≥ 80% | ≥ 80% | ≥ 80% |
| 2 x 150 bp | ≥ 75% | ≥ 75% | ≥ 75% | ≥ 75% |
| 2 x 250 bp | ≥ 75% | N/A | N/A | N/A |
| Run Time | ||||
| 2 x 50 bp | ~13 hr | ~13 hr | ~16 hr | N/A |
| 2 x 100 bp | ~19 hr | ~19 hr | ~ 25 hr. | ~36 hr |
| 2 x 150 bp | ~25 hr | ~25 hr | ~36 hr. | ~44 hr. |
| 2 x 250 bp | ~38 hr | N/A | N/A | N/A |
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:
- Minimize index hopping in indexed runs
- Effects of Index Misassignment on Multiplexing and Downstream Analysis
- Recommended strategies for unique dual index designs
- YouTube Video: Index Hopping Effects and Mitigation Strategies
Library Preparation
Sequencing library construction is available through the GSF using the Illumina sample preparation kits and Tecan sample preparation kit. The following library services are offered:
- Tecan Universal Plus mRNA-seq - Generate mRNA libraries directly from total RNA that provide strand of origin for sequenced mRNA Transcripts. Oligo dT capture based method. UDI adapters with primer-dimer free technology.
- Illumina 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. UDI adapters
- Illumina RNA Exome – Convert total RNA into template molecules of known strand of origin coupled with sequence specific probe capture of coding mRNA. Allows interrogation of low yield / low quality RNA.
- TruSeq Small RNA - Generate small RNA / miRNA libraries directly from total RNA.
- Illumina DNA Prep - Generate Libraries from Genomic DNA of limited quantity. UDI adapters
- TruSeq ChIP-Seq DNA - Selectively sequence DNA sequences bound by target proteins.
- Illumina DNA Prep for Exome Enrichment - Ideal for scalable exome sequencing studies.
- Tecan Ovation RRBS Methyl-seq w/ oxBS Module – Reduced Representation Bisulfite Sequencing (RRBS) is used to generate single base resolution DNA methylation information across a genomic sample. By analyzing a reduced representation of the genome, the amount of sequencing required is greatly reduced relative to whole genome bisulfite sequencing (WGBS). The TrueMethyl oxBS module allows the differential interrogation of both 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) and provides a method to accurately quantify the true level of cytosine methylation through the subtractive analysis of two parallel library sequence data sets, one with oxidative conversion of 5hmC and one without.
For other library constructs please inquire.