Design Content | Prepare Library | Sequence | Analyze Data

Data concordance between the NextSeq™
1000, NextSeq 2000, and NextSeq 550
Sequencing Systems
The latest NGS systems from Illumina offer innovative design while maintaining the same data
quality customers require for common sequencing applications.
Introduction
Over the last 20 years, Illumina has led the charge to improve nextgeneration sequencing (NGS) capabilities, making it easier and more
affordable to explore the genome, transcriptome, and epigenome. The
latest platforms from Illumina, the NextSeq 1000 and NextSeq 2000Sequencing Systems, offer breakthrough system design, chemistry
innovations, compatibility with an expansive list of library preparation
options, and onboard integrated informatics.
The NextSeq 1000 and NextSeq 2000 Sequencing Systems use thesame Illumina sequencing by synthesis (SBS) chemistry that powers
all Illumina platforms, including the NextSeq 550 System. Illumina SBS
chemistry is responsible for generating more than 90% of the world’s
sequencing data1 and delivers the highest yield of error-free reads2,enabling robust base calling across the genome.3
The NextSeq 1000 and NextSeq 2000 systems are optimized toincrease cluster brightness, reduce channel cross talk, and improvesignal-to-noise ratio. Combined with the latest advances in optics andinstrument design, the NextSeq 1000 and NextSeq 2000 Sequencing
Systems support increases in output and reduce the cost per run,all while maintaining the same high-quality data users have come toexpect from the NextSeq 550 System.
This application note demonstrates that the NextSeq 1000 andNextSeq 2000 Sequencing Systems deliver equivalent data quality to
the NextSeq 550 System for key applications, including exome, bulk,
and single-cell RNA gene expression.

with the NextSeq 500/550 High-Output Kit v2.5 (300 cycles) (Catalogno. 20024908), using a 2 × 100 bp run configuration. Eight samples
were multiplexed on a single run for each instrument.
Secondary data analysis was performed using the DRAGEN™
Enrichment Pipeline v3.4, available onboard the NextSeq 2000 Systemor through BaseSpace™ Sequence Hub. Variant calling accuracy wasassessed against the Platinum Genomes 2016 v1.0 data set.4

Bulk mRNA sequencing
Messenger RNA (mRNA) libraries were prepared from Universal
Human Reference RNA (Coriell Institute for Medical Research) using
the TruSeq™ Stranded mRNA Library Prep Kit (Illumina, Catalog no.
20020594).
Sequencing was performed on the NextSeq 2000 System withNextSeq 1000/2000 P2 Reagent Kit (200 cycles) using a 2 × 76 bp
run configuration. For comparison, the same libraries werealso sequenced on the NextSeq 550 System with the NextSeq
500/550 High-Output Kit v2.5 (300 cycles) using a 2 × 76 bp run
configuration. Twenty-four samples were multiplexed in a single run foreach instrument.
Secondary data analysis was performed using the DRAGEN RNA
Pipeline v3.5.112, available onboard the NextSeq 2000 System or in
BaseSpace Sequence Hub. Data were aligned against the Genome
Reference Consortium Human GRCh38 (h38 assembly).

Single-cell RNA sequencing

Methods
Exome sequencing
Exome libraries were prepared from NA12878 genomic DNA (gDNA)(Coriell Institute for Medical Research) using the Nextera™ DNA Flex
Pre-Enrichment Library Prep and Enrichment Reagents Kit (Illumina,Catalog no. 20025524) to capture genomic regions targeted by theIllumina Exome Panel (Illumina, Catalog no. 20020183).
Sequencing was performed on the NextSeq 2000* System with theNextSeq 1000/2000 P2 Reagent Kit (200 cycles) (Illumina Catalog no.
20040557), using the 2 × 100 bp run configuration. For comparison,
the same libraries were also sequenced on the NextSeq 550 System*While the NextSeq 2000 System has higher throughput than the NextSeq 1000 System,performance and data quality are equivalent. Therefore, only the NextSeq 2000 System
was used for data comparison studies in this application note.

For Research Use Only. Not for use in diagnostic procedures.

Samples for single-cell RNA sequencing (scRNA-Seq) were preparedfrom 4-plex peripheral blood mononuclear cells (PBMCs) isolated
from whole blood from a single donor. Libraries were prepared from~1000 PBMCs using the Chromium Single Cell Gene Expression v3Solution (10x Genomics, Catalog no. 1000092) on the ChromiumController (10x Genomics, Catalog no. 120223).
Sequencing was performed on the NextSeq 2000 System with
NextSeq 1000/2000 P2 Reagent Kit (200 cycles). For comparison,
the same libraries were also sequenced on the NextSeq 550 Systemwith the NextSeq 500/550 High-Output Kit v2.5 (150 cycles) (Illumina,
Catalog no. 20024907). Run configurations were set up according toparameters provided by 10x Genomics: 28-cycle read 1, 8-cycle index
read, and 91-cycle read 2.
Data analysis was performed using Cell Ranger scRNA-Seq analysis
pipelines (10x Genomics, Inc.) to align reads, perform clustering, andanalyze gene expression.

970-2020-001-B | 1

Design Content | Prepare Library | Sequence | Analyze DataResults
Exome sequencing
Primary and secondary analysis metrics, including precision and recallfor both single nucleotide variants (SNVs) and insertions/deletions(indels), run output (yield), error rate, % aligned reads, mean target
coverage depth, and uniformity of coverage, were evaluated. Boththe NextSeq 550 and NextSeq 2000 Systems exceeded publishedspecifications for sequencing output and data quality, with excellent
performance on both sequencing platforms (Table 1). These data
demonstrate that exome sequencing results on the NextSeq 2000
System are equivalent to the NextSeq 550 System, with both
platforms producing high-quality data and highly accurate variant
calling.
Table 1: Equivalent performance for exome sequencingNextSeq 550
SystemNextSeq 2000
System Run 1

NextSeq 2000
System Run 2

Primary run metrics
Clusters PF

514.2M

573.0M

565.1M

Yield

103.9 Gb

114.6 Gb

113.1 Gb

% Q30

92.20%

94.14%

94.15%

Error rate

0.34%

0.16%

0.16%

Autosome callability

95.61%

96.90%

96.90%% aligned reads
Mean target coverage
depth
Uniformity of coverage

99.15%

99.53%

99.50%

78.05

81.87

82.42

93.20%

96.92%

96.94%

SNV precision

99.3%

99.56%

99.59%

SNV recall

94.1%

94.88%

94.94%

Indel precision

88.0%

95.34%

95.71%

Indel recall

83.3%84.90%

84.97%

Secondary analysis metricsa

a. Averaged over 12 samples.

Bulk mRNA sequencing
Both the NextSeq 550 and NextSeq 2000 Systems exceeded
published specifications for sequencing output and data quality(Table 2). Quantification of specific RNA targets by bulk mRNA-Seq
showed excellent concordance between the two platforms (R2 > 0.99)
across four replicates (Figure 1). These data reinforce the findings thatthe NextSeq 2000 System produces data quality equivalent to theNextSeq 550 System for quantifying mRNA expression levels.

NextSeq 550
SystemNextSeq 2000System Run 1

NextSeq 2000
System Run 2

Clusters PF

422.4M

537.8M

531.4M

Yield

63.6 Gb

80.2 Gb

79.75 Gb

% Q30

92.85%

92.93%

92.28%

Error rate
Mean no. of genes
detected (n = 24)
Mean no. of transcripts
detected (n = 24)

0.27%

0.14%

0.16%

24,35526,507

24,73974,387

88,12581,948

2 | 970-2020-001-B

Single-cell whole-transcriptome sequencing
The NextSeq 550 and NextSeq 2000 Systems exceeded publishedspecifications for sequencing output and data quality (Table 3).
Quantification of individual cells using unique molecular identifiers(UMIs) in scRNA-Seq showed excellent concordance between thetwo platforms (R2 > 0.99) across four replicates (Figure 2). These data
further demonstrate that the NextSeq 2000 System produces data
quality equivalent to the NextSeq 550 System for scRNA-Seq studies.
Table 3: Equivalent performance for scRNA-Seq
MetricTable 2: Equivalent performance for bulk mRNA-Seq
MetricFigure 1: Highly concordant results for mRNA-Seq—Quantification of specific
RNA targets by bulk mRNA-Seq using the NextSeq 550 System are plotted onthe y-axis. Results using the NextSeq 2000 System are plotted on the x-axis.
Concordance of data is observed along the y = x trendline (R2 > 0.99 across fourreplicates).

NextSeq 550
SystemNextSeq 2000
System Run 1

NextSeq 2000
System Run 2

Clusters PF

454.8M

498.7M

530.1M

Yield

53.2 Gb

58.3 Gb

62.0 Gb% Q30 R1

96.28%

94.05%

93.91%% Q30 R2

87.73%

91.35%

91.51%

Error rate

0.29%

0.14%

0.14%

No. of genes detected
Median UMI counts
per cellFraction of reads in cells

18,52918,541

18,43852205175

5132

95.05%

94.95%

94.93%Estimated no. of cells

1181

1185

1184

For Research Use Only. Not for use in diagnostic procedures.

Design Content | Prepare Library | Sequence | Analyze Data

Summary
The NextSeq 1000 and NextSeq 2000 Sequencing Systems feature
breakthrough system design, chemistry innovations, and onboard
informatics that revolutionize what can be accomplished with abenchtop sequencing system. These platforms deliver improvements
in sequencing costs and run capacity while maintaining the same highquality data users expect from Illumina. Data from key applications
commonly run on the NextSeq 550 System, including exome, bulkmRNA, and single-cell RNA sequencing, were directly compared todata generated using the NextSeq 2000 System. Results show that
performance on both the NextSeq 550 and NextSeq 2000 Systems
is highly concordant and reflects the commitment of Illumina to deliverconsistent, high-quality sequencing performance reliably.

Learn more
To learn more about the NextSeq 1000 and NextSeq 2000
Sequencing Systems, visit www.illumina.com/systems/sequencingplatforms/nextseq-1000-2000.html
To download the data sets referenced in this note, visit
basespace.illumina.com/datacentral.

References
1.
2.
Figure 2: Highly concordant results for scRNA-Seq—Quantification ofindividual cells (UMIs) by scRNA-Seq using the NextSeq 550 System are plottedon the y-axis. Results using the NextSeq 2000 System are plotted on the x-axis.
Concordance of data is observed along the y = x trendline (R2 > 0.99 across fourreplicates).

3.
4.

Data calculations on file. Illumina, Inc. 2017.
Based on a comparison of the top three industry-leading NGS platforms.
Data calculations on file. Illumina, Inc. 2016.
Ross MG, Russ C, Costello M, et al. Characterizing and measuring bias insequence data. Genome Biol. 2013;14:R51.
Eberle MA, Fritzilas E, Krusche P, et al. A reference data set of 5.4 million
phased human variants validated by genetic inheritance from sequencing athree-generation 17-member pedigree. Genome Res. 2017;27:157–164.

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information, see www.illumina.com/company/legal.html. 970-2020-001-B QB10338

For Research Use Only. Not for use in diagnostic procedures.

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