HiSeq2000 was launched as a new sequencing instrument by Illumina, Inc. in 2010, with the same principle as Genome Analyzer, using a stable reversible terminator sequencing-by-synthesis method. The technology uses four kinds containing terminal blocking group and different fluorescent signal bases to complete complementary strand synthesis, not only to ensure the high accuracy and sequencing order, but also to exclude the sequencing error caused by the repeat sequences and the homopolymer. Unlike Genome Analyzer, HiSeq2000 combines the optical systems and manufacturing processes, uses two laser sources on the Flow Cell Scan, at the same time, four cameras on the four kinds of bases were recorded to reduce signal interference between different bases improved sequencing accuracy. HiSeq2000 adopts dual surface imaging technology, Flow Cell effective area increased, thereby increasing the throughput, reducing sequencing costs.  

Performance Parameters

Read Length

Single Flow Cell

Dual Flow Cell


Run Time


Run Time



1.5 days


2 days



4.5 days


5.5 days



8.5 days


11 days


2×50bp Q30≥85%*

2×100bp Q30≥80%*

*Install specifications for HiSeq sequencers with an Illumina PhiX library and cluster densities between 610-678 K/mm2 that pass filtering on a HiSeq system using TruSeq v3 Cluster and SBS kits for HiSeq. Performance may vary based on sample quality, cluster density, and other experimental factors.

Technical Features

1. Dual surface imaging technology;

2. Four cameras were taking pictures of four kinds of bases, reducing signal interference;

3. After HiSeq2000 v3 reagents upgrade, Flow Cell width of each lane widening, increased the Flow Cell area; Cluster generation reagents upgrade reduces occur of GC bias during sequencing.  

Sequencing Process


Field of application

1. Genomics

(1) De novo whole genome sequencing: high-output, high accuracy and low cost;

(2) Whole genome resequencing;

(3) Exome & target regions capture sequencing;

2. Transcriptomics

(1) Transcriptome sequencing

(2) Digital Gene Expression sequencing

(3) Small RNA Sequencing

(4) Degradation sequencing

3. The apparent genomics

(1) Methylation Sequencing

(2) RRBS Sequencing

(3) MeDIP Sequencing

(4) ChIP sequencing