RAPID RNA-SEQ LIBRARY PREP FOR SEQUENCING ON ILLUMINA® PLATFORMS
NEXTFLEX® Rapid RNA-Seq Kit for Illumina® Sequencing
- Faster than traditional Illumina® RNA library prep protocols
- Complete solution includes thermostable NEXTFLEX® Rapid Reverse Transcriptase
- Input – 10 ng – 1 µg total RNA for enrichment by NEXTFLEX® Poly(A) Beads 2.0 or ~ 1 ng – 100 ng isolated mRNA or rRNA-depleted RNA
- 96 barcodes are available for multiplexing
- Automation protocol is available for the Sciclone® NGSx workstation
- Functionally validated with Illumina® sequencing platforms
For research use only. Not for use in diagnostic procedures.
Multiplexing up to 96 RNA Libraries for Illumina® Sequencing
The patent pending NEXTFLEX® Rapid RNA-Seq Kits for Illumina® RNA-seq library prep provide an easy and flexible solution for generating single end or paired-end libraries as well as a variety of multiplexing options. This kit incorporates the NEXTFLEX® Rapid Reverse Transcriptase, a robust, thermostable RT that executes cDNA conversion with robust yields.
This kit was designed to be used in conjunction with the NEXTFLEX® RNA-Seq Barcodes or NEXTFLEX-96™ RNA-Seq Barcodes for multiplexing. The availability of up to 96 unique adapter barcodes makes this the most high-throughput kit available for RNA library prep.
Automated RNA Library Prep Protocols
An optimized automation protocol for RNA library prep using the NEXTFLEX® Rapid RNA-Seq Library Prep Kit are available for the Sciclone® NGSx workstations is now available. Download the Sciclone® NGS and NGSx Workstation Automation Guide for the NEXTFLEX® Rapid RNA-Seq Kit.
Magnetic Beads for mRNA Purification
The NEXTFLEX® Poly(A) Beads 2.0 now provide a convenient method for batch purification of pure, intact mRNA upstream NEXTFLEX® Rapid RNA-Seq library preparation. NEXTFLEX® Poly(A) Beads 2.0 use oligo(dT) primer to isolate polyadenylated messenger RNAs from 10 ng – 5 µg of previously isolated total RNA.
- NEXTFLEX® RNA Fragmentation Buffer
- NEXTFLEX® First Strand Synthesis Primer
- NEXTFLEX® First Strand Synthesis Buffer Mix
- NEXTFLEX® Rapid Reverse Transcriptase
- NEXTFLEX® Second Strand Synthesis Mix
- NEXTFLEX® Adenylation Mix
- NEXTFLEX® Ligation Mix
- NEXTFLEX® RNA-Seq Barcode Adapter 1 (0.6 μM)
- NEXTFLEX® Primer Mix (12.5 μM)
- NEXTFLEX® PCR Master Mix
- Nuclease-free Water
- Resuspension Buffer
REQUIRED MATERIALS NOT PROVIDED
- 10 ng – 1 µg total RNA for enrichment by NEXTFLEX® Poly(A) Beads 2.0 or ~ 1 ng – 100 ng isolated mRNA or rRNA-depleted RNA
- DynaMag™-2 Magnet (Life Technologies Cat # 123-21D)
- Commercial kits for rRNA depletion (optional, if not using Poly(A) enrichment)
- 100% Ethanol (stored at room temperature)
- 80% Ethanol (stored at room temperature)
- 2, 10, 20, 200 and 1000 μL pipettes
- RNase-free pipette tips
- Nuclease-free 1.5 mL microcentrifuge tubes
- Thin wall nuclease-free 0.5 mL microcentrifuge tubes
- 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
- Adhesive PCR Plate Seal (Bio-Rad, Cat # MSB1001)
- Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63881)
- Magnetic Stand -96 (Thermo Fisher®, Cat # AM10027) or similar for post PCR cleanup
- Heat block
Streamlined Library Construction for Quantitative, Directional, and Standard RNA-Seq
RNA Sequencing (RNA-Seq) is a valuable tool for a broad range of clinical, environmental, and basic research. Producing high quality RNA-Seq libraries can be challenging for several reasons, including isolation of pure RNA, efficiently converting RNA to cDNA, and loss of material incurred during the series of enzymatic steps and cleanups required for library construction. Here we introduce PerkinElmer’s family of NEXTFLEX® Rapid RNA-Seq kits for Illumina® sequencing, all of which include the thermostable NEXTFLEX® Rapid Reverse Transcriptase enzyme. The NEXTFLEX® Rapid RNA-Seq Kits provide affordable and unique technology, some of the shortest RNA-Seq library preparation times of any kits on the market, include all enzymes required for library preparation, and produce the highest quality RNA-Seq library.
The NEXTFLEX® Rapid RNA-Seq Kits allow the end user to complete library construction in less than 1 day. In contrast, the previous generation of NEXTFLEX® RNA-Seq Kits, as well as the current Illumina® TruSeq® RNA-Seq Kits, require over 7 hours for completion (Table 1). By combining second strand synthesis and end repair into the same reaction, the need for a separate end repair step and cleanup is eliminated. This improvement is time efficient and reduces loss of material during cleanup.
Table 1. Comparison of RNA-Seq library preparation kit protocols and time to completion. Note that times include bead cleanups and account for time required from RNA fragmentation to the final bead cleanup after PCR.
|Kit||Time to Completion||Steps Required||Bead Cleanups Required||Reverse Transcriptase Included?|
|NEXTFLEX® Rapid RNA-Seq Kit||5 hrs 50 mins||6||3||Yes|
|Illumina® TruSeq® RNA V2||7 hrs 10 mins||7||4||No|
Thermostable NEXTFLEX® Rapid Reverse Transcriptase for Improved First Strand Synthesis and Library Yield
A critical step in RNA-Seq library construction is the conversion of RNA to cDNA. All NEXTFLEX® Rapid RNA-Seq Kits include NEXTFLEX® Rapid Reverse Transcriptase (RT) enzyme, in contrast to our previous RNA-Seq kits as well as to the Illumina® TruSeq® kit (Table 1). The NEXTFLEX® Rapid RT is a thermostable, RNaseH minus enzyme that functions optimally at 50˚ C, a higher temperature than standard Moloney-Murine Leukemia Virus (M-MLV) reverse transcriptases, which function at 42˚ C. This elevated temperature allows for reduced secondary structure in RNA templates and therefore increased efficiency of first strand synthesis.
To examine the efficacy of NEXTFLEX® Rapid RT in library construction, we compared library yields using cDNA produced by different enzymes. The same pool of Poly (A)+ selected mRNA isolated from murine Ag8 cells was used in first strand synthesis with either NEXTFLEX® Rapid RT or SuperScript® III (Figure 1). Higher library yields were obtained using the NEXTFLEX® Rapid RT coupled with the NEXTFLEX® Rapid RNA-Seq Kit. These results demonstrate improved library yields as a result of optimized first strand synthesis using NEXTFLEX® Rapid RT.
Figure 1. Improved library yield using NEXTFLEX® Rapid RT. High Sensitivity DNA Bioanalyzer traces of RNA-Seq libraries constructed with NEXTFLEX® Rapid RNA-Seq Kits. Libraries were constructed using 10 ng of fragmented, Poly (A)+ mRNA converted to cDNA using either NEXTFLEX® Rapid RT (blue) or SuperScript® III (red).
High-quality RNA-Seq Data
Further analysis of NEXTFLEX® Rapid RNA-Seq library quality was carried out using Illumina® sequencing, as library yield is only one metric of library quality. Libraries were prepared with the NEXTFLEX® Rapid RNA-Seq Kit using 10 ng aliquots of a single murine Ag8 cell Poly (A)+ RNA sample, so as to disentangle biological variation from technical variation. Libraries were sequenced on an Illumina® HiSeq® 2500 instrument using a 67 bp single end RAPID run. Resulting reads were trimmed based on a quality score moving window using sickle and mapped to the UCSC mm10 assembly using TopHat 2.0.10. We obtained a total of 31,949,336 reads for Rapid RT libraries and 31,720,922 for Superscript III libraries (Table 2). For transcript representation purposes, only reads mapping to exons, specifically all annotated 5’ UTRs, coding sequences, and 3’ UTRs, were further considered. The number of total reads as well as unique reads mapping to exons were similar between the two enzymes; however, a greater number of transcripts, 12,418 vs. 12,129, were represented in the NEXTflex Rapid RT libraries vs. SuperScript III libraries, respectively.
Table 2. Read counts in NEXTFLEX® Rapid RNA-Seq libraries and unique reads after mapping to exons.
|Rt Enzyme||NEXTFLEX® Rapid RT||Superscript® III RT|
|Reads After Quality Trimming||31,891,608||31,663,364|
|Unique Reads Mapping to Exons||17,411,368||17,487,086|
|Unique Reads in Exons (%)||54.60||55.23|
|Number Transcripts Represented||12,418||12,129|
A further analysis of mapped reads was performed. Read quality before and after mapping was visualized using FastQC and GC content as a function of reads examined (Figure 2). Read quality is excellent in the total library and is slightly higher in the remaining set of trimmed, mapped reads, which is expected given a quality-aware trimming step was carried out. Furthermore, GC content is very similar to the theoretical distribution, indicating accuracy in transcript representation. Visual inspection of several genes demonstrates read coverage across all exons (Figure 3A and B). Furthermore, a metagene analysis of read coverage across all genes divided into 100 bin segments, demonstrates even read coverage across the 5’-, gene body, and 3’-ends of transcripts (Figure 3C).
Figure 2. Metrics of NEXTFLEX® Rapid RNA-Seq data determined by FastQC. Quality score plots for (top) and mean GC content (bottom) for (A) all reads or (B) reads mapping to exons. Plots shown correspond to NEXTFLEX® Rapid RNA-Seq libraries constructed using NEXTFLEX® Rapid RT.
Figure 3. Read coverage across gene bodies. Mapped reads at the (A) Trp53 and (B) Eif5b loci scaled to read density as indicated. (C) Metagene plot of read density across all annotated loci. All gene bodies and mapped read densities are scaled to 100 bin segments; mean read density is shown in reads per kilobase per million mapped reads (RPKM; solid line) +/- standard error across replicates (faded bands). Shown is read signal corresponding to libraries made with either NEXTFLEX® Rapid RT (red) or SuperScript III (blue).
Finally, we examined library consistency across protocols. We compared RNA-Seq read counts in exons from libraries constructed using the NEXTFLEX® Rapid RNA-Seq Kit to longer traditional protocols. Indeed, libraries constructed using the two methods were highly similar (r = 0.9976; Figure 4). These data demonstrate that the NEXTFLEX® Rapid RNA-Seq protocol provides significantly faster library construction with enhanced reverse transcriptase performance.
Figure 4. Correlation between Rapid and Standard RNA-Seq data. Pearson’s correlation between the average log2 of normalized counts per million (CPM) of all genes across all replicates of Rapid RNA-Seq vs. Standard RNA-Seq, r = 0.9976.
The present study demonstrates the utility of the NEXTFLEX® Rapid RNA-Seq Kits as a method to improve speed of library preparation without any compromise in library quality. All NEXTFLEX® Rapid RNA-Seq Kits include NEXTFLEX® Rapid RT, a high performance thermostable enzyme, thus providing all components required for library construction within a single kit. NEXTFLEX® Rapid RNA-Seq Kits produced high-quality sequencing data with improved transcript representation. In addition, the increased library yield using NEXTFLEX® Rapid RT is an important improvement for users with low-input amounts. For researchers wishing to multiplex libraries, PerkinElmer continues to offer our full range of up to 96 NEXTFLEX® RNA-Seq adapter barcodes. The NEXTFLEX® Rapid RNA-Seq and Rapid Directional RNA-Seq kits provide a streamlined workflow for users to create high-quality RNA-Seq libraries with less hands-on time.
5’ to 3’ Sequence Coverage
Normalized coverage by position. For each library, the average coverage is shown at each relative position along the transcripts’ length.
Selected Publications that Cite the Use of the NEXTFLEX Rapid RNA-Seq Kit:
- Boutrin, M.-C., et al. (2015) A putative TetR regulator is involved in Nitric Oxide stress resistance in Porphyromonas gingivalis. Molecular Oral Microbiology. doi: 10.1111/omi.12128.
- Páneka, T., et al. (2016) First multigene analysis of Archamoebae (Amoebozoa: Conosa) robustly reveals its phylogeny and shows that Entamoebidae represents a deep lineage of the group. Molecular Phylogenetics and Evolution. doi:10.1016/j.ympev.2016.01.011.
- Park, S. J., et al. (2014) Optimization of crop productivity in tomato using induced mutations in the florigen pathway. Nature Genetics. 46, 1337–1342. doi:10.1038/ng.3131.
- Tan, M. H., et al. (2015) First comprehensive multi-tissue transcriptome of Cherax quadricarinatus (Decapoda: Parastacidae) reveals unexpected diversity of endogenous cellulose. Organisms Diversity & Evolution. Pg 1 – 16. Doi: 10.1007/s13127-015-0237-3.