Specifications for the MiSeq i100 Series Instruments

Output per flow cell for various read lengths****a

What is read length? Sequence read length refers to the length of each sequenced DNA fragment and whether it is sequenced from one end (single read) or both ends (paired-end reads). For example, a 2 × 150 bp run generates two reads (forward and reverse) of 150 base pairs for each DNA fragment. Longer read lengths have a better probability of spanning repeats in the DNA sequence, while shorter read lengths can be cost-effective for counting methodologies where broad genome coverage is not required.

Table 1. Yield Expectations Based on Read length.

Run Output	Flow Cell Type
	5Mb	25Mb	50Mc	100Mc
1 x 100bp (100 cycle)	-	2.5 Gb	5 Gb	10 Gb
2 x 150 bp (300 cycle)	1.5 Gb	7.5 Gb	15 Gb	30 Gb
2x 300 bp (600 cycle)	3 Gb	15 Gb	30 Gb	-

1. Specifications based on Illumina PhiX control library at supported cluster densities.

2. 5M and 25M flow cells available with platform release.

3. 50M and 100M flow cells will be available starting in 2025 for the MiSeq i100 Plus System only.

Reads passing filter per flow cell

What are reads passing filter(PF)? The percentage of clusters passing chastitya filtering, with each passing filter cluster providing a read (single-end reads) or two reads (paired-end reads). Clusters pass filter if no more than one base call has a chastity value below 0.6 in the first 25 cycles. Single-end runs involve sequencing DNA from only one end and offer an economical alternative. Paired-end runs sequence both DNA ends for easier analysis of rearrangements, novel transcripts, and more.

Table 2. Read Specifications

Reads PF per flow cell	5Mb	25Mb	50Mc	100Mc
Single Reads PF	5M	25M	50M	100M
Paired-End Reads PF	10M	50M	100M	200M

1. Chastity is defined as the ratio of the brightest base intensity divided by the sum of the brightest and second brightest base intensities.

2. 5M and 25M flow cells available with platform release.

3. 50M and 100M flow cells will be available starting in 2025 for the MiSeq i100 Plus System only.

Quality scores****a

What is a quality score? A quality score, or Q score, is a prediction of the probability of an error in base calling. Higher Q scores indicate a smaller probability of error. A quality score of 30 represents an error rate of 1 in 1000 with a corresponding call accuracy of 99.9%.

Table 3. Q30 Score Specifications

Run Configuration	Quality Scores
1 x 100bp (100 cycle)	≥ 90 % of bases higher than Q30
2 x 150 bp (300 cycle)	≥ 90 % of bases higher than Q30
2x 300 bp (600 cycle)	≥ 85 % of bases higher than Q3

1. The percentage of bases ≥ Q30 is averaged across the entire sequencing run. Quality scores are based on MiSeq i100 Series reagents using an Illumina PhiX control library.

2. 5M and 25M flow cells available with platform release.

3. 50M and 100M flow cells will be available starting in 2025 for the MiSeq i100 Plus System only.

Run time

What is run time? Sequencing run time includes cluster generation, onboard denaturation, sequencing, base calling, and quality scoring on the instrument.

Table 4. Run Time Estimates

Run Configuration	Quality Scores
1 x 100bp (100 cycle)	≥ 90 % of bases higher than Q30
2 x 150 bp (300 cycle)	≥ 90 % of bases higher than Q30
2x 300 bp (600 cycle)	≥ 85 % of bases higher than Q3

Estimated sample throughput for key applications****a

What is sample throughput? Sample throughput is the number of samples that can be sequenced in a single run per flow cell.

		Number of samples per flow cell
Application	Reads per sample	5Mb	25Mb	50Mc	100Mc
Transcriptomics
3’ gene expression	1−5M	1−5	5−25	10−50	25−100
Targeted RNA panel	1−5M	1−5	5−25	10−50	25−100
mRNA-Seq	10−25M	—	1−2	1−5	1−10
Total RNA-Seq	50M	—	—	1	1−2
Microbial genomics
Pathogen detection	0.5−1M	1−10	1−50	1−100	1−200
16S amplicon sequencing	0.1−0.2M	1−50	1−250	1−384	1−384
Shallow shotgun metagenomics	0.5−10M	1−10	1−12	1−25	1−50
Shotgun metagenomics	10−25M	—	1−2	1−5	1−10
Small WGSd	0.5M	1−10	1−50	1−100	1−200
Targeted gene sequencing
Amplicon-based	0.1−50M	1−5	1−250	1−384	1−384
Enrichment-based	0.1−50M	1−50	1−250	1−384	1−384
Genome editing	0.1−50M	1−50	1−250	1−384	1−384
Immune repertoire	2−25M	—	1−12	1−25	1-50
Quality control
Library QC	> 0.02M	up to 384-plex	up to 384-plex	up to 384-plex	up to 384-plex

1. Reads per sample and sample throughputs are estimates and highly variable, depending on the panel and desired coverage.

2. 5M and 25M flow cells available with platform release.

3. 50M and 100M flow cells will be available starting in 2025 for the MiSeq i100 Plus System only.

4. Estimated reads per sample based on minimum 30× coverage of an average 5MB bacterial genome.

For any feedback or questions regarding this article (Illumina Knowledge Article #9204), contact Illumina Technical Support [e-mail].