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

Output

Run Time

Output

Run Time

1×35

47-52Gb

1.5 days

95-105Gb

2 days

2×50

135-150Gb

4.5 days

270-300Gb

5.5 days

2×100

270-300Gb

8.5 days

540-600Gb

11 days

Performance

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

 

HiSeq2500 (the upgraded version of Illumina HiSeq2000 sequencing instrument) was launched in 2012. There are 20 Hsieq2500 in BGI and distributed around the world. Compared with HiSeq2000, HiSeq2500 has two sequencing modes: High Output mode and Rapid mode. High Output mode has the same pattern as HiSeq2000 sequencing instrument; there is no change in sequencing reagents and Flow Cell. Rapid mode uses the new Flow Cell and sequencing reagents, shortening sequencing time, sequencing read lengths up to 150bp.

Performance Parameters

Read Length

High Output Mode*

Rapid Mode*

Dual Flow Cell

Single Flow Cell

Dual Flow Cell Run Time

Dual Flow Cell

Single Flow Cell

Dual Flow Cell Run Time

1×36

95-105Gb

47-52Gb

2 days

18-22Gb

9-11Gb

7 hours

2×50

270-300Gb

135-150Gb

5.5 days

50-60Gb

25-30Gb

16 hours

2×100

540-600Gb

270-300Gb

11 days

100-120Gb

50-60Gb

27 hours

2×150

--

--

--

150-180Gb

75-90Gb

40 hours

Performance

2×50bp Q30≥85%

2×100bp Q30≥80%

2×50bp Q30≥85%

2×100bp Q30≥80%

2×150bp Q30≥75%

*Install specifications based on Illumina PhiX control library at supported cluster densities (between 610-678 K clusters/mm2 passing filter using TruSeq v3 or 700-820 K clusters/mm2 passing filter using TruSeq Rapid kits). Run times for rapid run mode correspond to onboard cluster generation(1.5 hours) and sequencing; for high-output mode, run times correspond to sequencing only, not include Flow Cell Preparation time(4-5hours). Performance may vary based on sample quality, cluster density, and other experimental factors. HiSeq2000 instruments prior to serial number 700895, rapid mode to extend the running time of approximately 15 hours when upgraded.

Technical Features

1. Rapid mode uses 2 lane Flow Cell and new sequencing reagents, sequencing time is shortened to a few hours, the sequencing read lengths up to 150bp;

2. Dual flow path, supporting high output and rapid sequencing modes;

3. Deselect lanes to bypass image process, reduce scan time

4. Complete 1 human genome sequencing in one day.  

Sequencing Process

High Output mode:

 Rapid mode:


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

Miseq (Illumina) is a new generation of miniaturized sequencing instrument (bench-top) launched on February 2011, which uses a chemical method based on Illumina TruSeq technology that nucleotide sequencing-by-synthesis by reversible terminator. Compared with Hiseq2000, the unparalleled accuracy of next-generation sequencing reagents, transitory sequencing time by   new fluid system are the main advantages, although the throughput of a run is low.

 

Performance Parameters

Read Length

Run Time

Throughput

Quality

2×150bp

About 24 hours

4.5-5.1Gb

Q30>80%

2×250bp

About 39 hours

7.5-8.5Gb

Q30>70%

Explanation:

(1)The above performance is based on Miseq sequencing V2 reagent. V1 reagent only supports 2x150bp, runs about 27 hours, 1.5-2Gb throughput, Q30>80%. But V1 reagent is no longer ordered.

(2)This data is based on Illumina control libraries PhiX (a balanced representation of A, T, G, and C nucleotides). The appropriate clusters density is 900-1200K/mm2, and 880-965K/mm2 after PF. But different sample sequencing results by their own properties.

 

Technical Features

1.    Short sequencing cycle: Miseq V2 150PE is short to 24 hours running time, so it can used for fast and efficient amplicon sequencing and small genome sequencing, with a small amount of time to complete projects of small amount of data.

2.    Up to 250bp of paired-end read-length:Miseq sequencing read length has reached 2x250bp, which can effectively across complex genomes highly repetitive regions,thereby enhancing the effect of assembly, more conducive for gene annotation and gene function dig.

3.    Convenient sequencing process: Miseq is a compact, integrated platform for integration of cluster generation, Pair-end sequencing and complete data analysis, saving laboratory space. Through an intuitive touch-screen interface to carry out simple instrument operation, plug and play reagent with RFID tracking, with automated convenience.

 

Sequencing Process



Applications

1.Amplicon sequencing

Long read-length capability of paired-end, speed and high data quality, so ampilicon sequencing project can be completed within a few days.

2.Transcriptome sequencing

Compared with the 454 sequencer, similar length,cheap,easy to implement de novo assembly, more comprehensive gene annotation.

3.Small genomes De novo assembly

2x250bp read length and high-throughput up to 7G are suitable as the project for small genome such microbes because of mosaic effect.

4.Microbial diversity analysis

2x250bp read length can measured through some of the variable region, more conducive to improving the accuracy of analytical results.