The Illumina HiSeq 2500 allows for extremely high throughput and deep analysis of genomes and transcriptomes for relatively low cost.
Two operation modes can be run on the HiSeq 2500 depending on data output and speed:
High-Output Mode (run duration: 5-11 days)
In high-output (HO) mode, a single lane of sequencing can generate 180-210 million reads, with read lengths of 50 and 100 bp (for a potential output of 10 billion to 20 billion base pairs of sequence). Both ends of fragments can also be sequenced and tracked, called paired-end sequencing (PE), which is useful for assembly of new genomes or transcriptomes. A flow cell in HO mode is composed of 8 lanes, for a potential of around 300 billion or more bases sequenced in a single run.
Rapid Read Mode (run duration: 16-48 hours)
A lane in Rapid Read (RR) mode delivers about 20% less data than HO mode and costs about 20% more, but results can be obtained in one or two days, as opposed to 5-11 days. In RR, one pays more for rapid generation of data, normally both lanes of the Rapid flow cell are purchased by a single user. Each lane of a two-lane RR flow cell can generate 130-150 million reads with read lengths of 50, 100, 150, or 250, single or paired end. Note that read lengths of 150 - 250 bp can be achieved in RR, not possible in HO.
The Illumina MiSeq allows for more targeted sequencing of smaller genomes and amplicons
MiSeq (run duration: 8-50 hours)
Uses a single lane flow cell and can produce 15-25 million reads per run with read lengths up to 300 bp, single or paired end. This sequencing system is good for metagenomic studies. MiSeq runs can also serve as a way of quality checking library preparations prior to running on the HiSeq.
MiSeq has a smaller output flow cell producing 1 million (MiSeq Nano) or 4 million (MiSeq Micro) reads, which can be used for sequencing library QC and small amplicon sequencing projects.
Applications and Recommended Run Types
1. Whole Genome Sequencing and Re-Sequencing (DNA-seq):
- Genome re-sequencing: mutant identification, evolutionary comparisons, disease tissue sequencing:
- Single read 100 (SR100) or paired end 100 (PE 100)
- De novo genome assembly for new unsequenced species:
- PE100 or PE150
2. Expression/Transcriptome Analysis (RNA-seq):
- De novo transcriptome analysis in an unsequenced or unannotated genome:
- PE100 or PE150
- RNA-seq to study comparative transcriptomics in model genomes (e.g. sequenced and annotated genomes):
- SR50 or SR100
- Small RNA transcriptomics:
3. Epigenomics and Gene Regulation:
- Chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factor interaction sites:
- SR50 or SR100
- Methylated DNA sequencing to identify methylated regions of the genome:
- SR100, PE100 or PE150 depending on the extent of annotation of the reference genome
3. Metagenomics, Amplicon, or Targeted Gene Sequencing:
- V2 and V3 reagents produce 15-20 million reads - good for Metagenomic or Targeted Gene sequencing projects
- Micro reagents produce 4 million reads - Nano reagents produce 1 million reads
- good for QC of custom library preps or small scale amplicon sequencing projects
Note: Users can download the Illumina Sequencing Coverage Calculator to estimate the number of lanes of sequencing required for your experiment.