- The library prep portfolio supports transcriptome-wide analysis, providing the flexibility to address the challenges of different species, sample types, and input volumes
- Scalable sequencing throughput to support a wide range of RNA-Seq applications
- Integrate DRAGEN secondary analysis to optimize workflow efficiency
Brief introduction
The NextSeq 1000/1000-CN and NextSeq 2000/2000-CN RNA Sequencing (RNA-Seq) Solutions (collectively, the "NextSeq 1000/2000 RNA-Seq Sequencing Protocols") provide a clear, complete view of the transcriptome, making it easier than ever. The solution is powered by Illumina's cutting-edge next-generation sequencing (NGS) technology and optimized sequencing-by-synthesis (SBS) chemistry, XLEAP-SBS™ chemistry, which can be integrated from library preparation to data analysis (Figure 1). The flexibility and scalability of the NextSeq 1000/1000-CN and NextSeq 2000/2000-CN Series Sequencing Systems (collectively, the "NextSeq 1000/2000 Series Sequencing Systems") (Figure 2) enable users to efficiently handle a wide range of sample throughput, ensuring the ideal balance between sequencing budget and sample throughput. The NextSeq 1000/2000 RNA-Seq Solution supports a range of RNA applications, from basic gene expression profiling to complex whole transcriptome analysis.
Figure 1: NextSeq 1000/2000 RNA-Seq Workflow NextSeq 1000/2000 Series Sequencing Systems – A simple, integrated NGS sequencing protocol that provides highly accurate RNA-Seq sequencing data. Working hours vary depending on the type of experiment and assay.
Figure 2: NextSeq 1000/2000 Series Sequencing Systems—NextSeq 1000/2000 Series Sequencing Systems are equipped with XLEAP-SBS chemistry and an on-board secondary data analysis module to integrate sequencing workflows.
Advantages of RNA-Seq
RNA-Seq is emerging as a method of particular choice for industry pioneers1,2 to present a detailed snapshot of the transcriptome at a specific point in time. There are many advantages over quantitative PCR, including:
- Hypothesis-free experimental design without prior knowledge of the transcriptome
- Greater exploratory capabilities for detection of known and novel transcripts
- Higher throughput capability to quantify hundreds to thousands of regions per assay
- The wider dynamic range, the more accurate the detection of gene expression
- More data per test, providing full sequence and variant information
Integrated NGS workflows
The NextSeq 1000/2000 Series Sequencing Systems support a wide range of library preparation solutions to meet the needs of a wide range of transcriptome studies. Researchers can choose from a variety of library preparation kits that best suit their experimental needs, helping them overcome common challenges such as poor starting RNA quality or limited sample volumes.
State-of-the-art Illumina RNA library preparation
With extensive experience in the field of RNA-Seq, Illumina offers proven solutions for RNA library preparation that we can trust. Illumina's portfolio of RNA Library Prep Kits continues to evolve to provide researchers with the high-quality data they need, with an integrated workflow that helps researchers complete all experiments in a standard work shift. Illumina offers three RNA library preparation kits (Table 1):
Table 1: Illumina RNA Library Prep Kit
a. Equipped with the Ribo-Zero Plus to remove large amounts of RNA from samples from multiple species, including human, mouse, rat, bacterial, and epidemiological samples.
b. 仅适用于人类样本。 使用Illumina Exome Panel 和Respiratory Oligos Panel v2 进行测试。 Illumina RNA Prep with Enrichment 不提供链信息。
c. 显示的为高质量RNA 的最低起始量。 为获得理想质量以及对于FFPE 样本，Illumina Stranded Total RNA Prep 的最低建议量为10 ng。
d. Manual operation and total time are based on the time required to manually process 24 samples (for Illumina Stranded Total RNA and mRNA workflows) and 1 sample (for enrichment workflows).
NextSeq 1000/2000
Serial sequencing system
The NextSeq 1000/2000 Series Sequencing Systems are powerful and flexible enough to meet the needs of a wide range of transcriptome profiling. The four flow cell types available allow researchers to choose the ideal balance between sample size and reads for each sample (Table 2). For example, gene expression profiling, which measures gene-level abundance of known traits, can be performed efficiently in high-throughput mode, with up to 170 sample † detected in a single run. Whole transcriptome analysis has the ability to discover new features, detecting coding and non-coding RNA from up to 34 samples per run, and researchers can detect up to 68 samples per run when analyzing coding RNA (Table 2, Table 3).
Table 2: Illumina RNA-Seq solution and sample throughput per flow cell
a.针对Illumina Stranded Total RNA Prep 和Illumina Stranded mRNA Prep，推荐读长为2 × 75 bp；针对Illumina RNA Prep with Enrichment，推荐读长为2 × 100 bp。
b. Illumina Stranded mRNA Prep 与FFPE 样本不兼容。 对于低质量样本或FFPE 样本，建议使用Illumina RNA Prep with Enrichment 或Illumina Stranded Total RNA Prep。
c. P3 和P4 试剂仅适用于NextSeq 2000/2000-CN 基因测序仪。
d. The NovaSeq X Plus Gene Sequencer supports single or dual flow cell operation. The NovaSeq X Gene Sequencer supports single-flow cell operation.
Up to 384 unique dual-label sequences are available. For the NovaSeq X Series, independent channel loading enables multiplexing of more samples.
表3：NextSeq 1000/1000-CN 和NextSeq 2000/2000-CN 进行RNA-Seq 的性能参数
a. Output specifications are based on a single flow cell, calculated using an Illumina PhiX control library at supported cluster densities.
Run time includes cluster generation, sequencing, and base calling on the NextSeq 1000/2000 Series Sequencing System.
c. Mass score calculations are based on Illumina PhiX quality control libraries. Performance may vary depending on library type and quality, insert size, loading concentration, and other experimental factors. Base ratios above Q30 are based on the average of the entire run.
xleap-SBS reagents for P1, P2, and P3 flow cells are available in Q2 2024.
e. P3 and P4 reagents are only available on the NextSeq 2000/2000-CN Series Sequencing System.
The NextSeq 1000/2000 Series Sequencing Systems provide cross-application flexibility, allowing researchers to easily transition between multiple sequencing projects (Figure 3). The system is compatible with a range of library preparation kits from Illumina and third-party, enabling easy conversion between population cell RNA-Seq, single-cell RNA-Seq, exome sequencing, and other applications. For example, researchers can combine RNA-Seq with exome sequencing on the NextSeq 1000/2000 Series Sequencing Systems to assess whether coding variants affect transcript expression, or perform ATAC-Seq‡ analysis of chromatin accessibility to better characterize functional regulation.
Explore with XLEAP-SBS chemistry
With the NextSeq 1000/2000 Series Sequencing System, researchers can drill down with higher read depths, more accurate fold change estimates, and higher sensitivity when detecting genes, transcripts, and differential expression. Powered by XLEAP-SBS chemistry, Illumina's fastest, highest-quality, and most reliable sequencing chemistry to date, the Nextseq 1000/2000 Series Sequencing Systems are the highest-throughput, lowest-cost single-sample sequencing systems of any Illumina benchtop sequencer.
† 表达谱分析假设每个样本10M reads。
‡ ATAC-Seq，转座酶可及性染色质测序分析。
NextSeq 1000/2000 Series Sequencing Systems reduce the cost per gigabyte of sequencing output, which, together with additional sequencing capabilities, helps:
- Each sample gets more reads to capture information about low-abundance transcripts
- More samples can be sequenced within a given study budget, allowing for more diversity in experimental design
- Capture the more complex aspects of RNA research with a more holistic approach that drives more discoveries
The dual-throughput mode of the NextSeq 1000/2000 Series Sequencing Systems (Table 3) allows researchers to optimize study designs based on sample volume and throughput needs. For example, the NextSeq 2000/2000-CN P4 flow cell provides additional sequencing capabilities, making RNA-Seq a routine tool in many laboratory molecular toolsets and providing more flexibility in experimental design. If higher sample throughput is required, the NovaSeq™ 6000 System can be used for research that scales to hundreds of samples per run (Table 2).
The value of paired-end sequencing
With the NextSeq 1000/2000 Series Sequencing System, researchers can perform single- or double-ended sequencing. Single-ended sequencing is cost-effective and suitable for gene expression profiling. However, paired-end RNA-Seq has key advantages. Transcriptional isoforms can be effectively distinguished by sequence depth information generated at both ends of the insert, allowing for more accurate detection and quantification of transcriptional-level abundance. Paired-end information significantly increases the sensitivity of detecting gene fusions as well as insertion/deletion (INDEL) variants.
Illumina's simplified analytics solution
DRAGEN™ secondary analysis
RNA-Seq data analysis can be performed using the tools in Illumina DRAGEN Secondary Analysis, which is an accurate, comprehensive, and efficient data analysis pipeline. §The Illumina DRAGEN RNA pipeline obtains data output from the NextSeq 2000/2000-CN sequencing system, enabling accurate RNA alignment to a reference genome, variant calling and quantification of genes, and characterization of splice sites and candidate gene fusions (Figure 3). The DRAGEN RNA pipeline can be performed on the NextSeq 1000/2000 Series Sequencing Systems using a DRAGEN hardware carrier.
§ NextSeq 1000/2000 Series Sequencing Systems include DRAGEN hardware. The instrument comes with a DRAGEN license and does not need to be purchased separately.
Figure 3: DRAGEN RNA Pipeline—An example of a screenshot of a differential expression heatmap using the DRAGEN RNA pipeline, which can be used on the NextSeq 1000/2000 Series Sequencing System
Powerful, fast, and flexible, the NextSeq 1000/2000 RNA-Seq Sequencing Protocol combines the NextSeq 1000/2000 Series Sequencing Systems with an advanced portfolio of RNA library preparation products and user-friendly RNA-Seq software applications to provide an integrated workflow from RNA library preparation to analysis of sequencing results. This sequencing protocol offers four flow cell configurations to ensure cost-effectiveness across a wide range of RNA-Seq project types.
- Geraci F, Saha I, Bianchini M. Editorial: RNA-Seq Analysis:Methods, Applications and Challenges. Front Genet.2020;11:220.doi:10.3389/fgene. 2020.00220
- Corchete LA, Rojas EA, Alonso-López D, De Las Rivas J,Gutiérrez NC, Burguillo FJ. Systematic comparison and assessment of RNA-seq procedures for gene expression quantitative analysis. Sci Rep. 2020; 10(1):19737.doi:10.1038/s41598-02076881-x
For research use only and not for diagnostic use.