GEL-FREE OR LOW INPUT SMALL RNA LIBRARY PREP KIT WITH REDUCED BIAS FOR ILLUMINA® SEQUENCING
NEXTFLEX® Small RNA-Seq Kit v3 for Illumina® Platforms
- Completely gel-free protocol with normal input amounts
- Greater discovery/detection rates reduce sequencing cost
- Randomized adapters reduce ligation bias, resulting in more accurate data
- Low input (1 ng) gel-based protocol included
- AIR™ Ligase offers greater sequencing depth
- Use more of your RNA sample – 10.5 µL input volume
- Barcoded primers are included in the kit allowing multiplexing of up to 48 samples
- An automation protocol is available for the PerkinElmer® Sciclone® G3 NGS/NGSx and the Zephyr® G3 NGS workstation
- Functionally validated with Illumina® sequencing platforms
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Perspectives on Small RNA Sequencing: Challenges & Complications of Small RNA Library Prep
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For research use only. Not for use in diagnostic procedures.
Gel-Free Small RNA Library Prep with Randomized Adapters for Reduced Bias
The NEXTFLEX® Small RNA-Seq Kit v3 uses patented and patent-pending technology to provide a reduced-bias small RNA library preparation solution for Illumina® sequencing platforms with gel-free or low-input options. Our approach to reducing ligation-associated bias involves the use of adapters with randomized bases at the ligation junctions, resulting in greatly decreased bias in comparison to standard protocols. This reduction in bias results in data that more accurately represents abundances of small RNAs in the starting material. In addition, reduction of bias allows more miRNAs to be detected with fewer total reads, increasing efficiency and reducing cost for small RNA sequencing.
PAGE purification, required for traditional small RNA library prep, is tedious, time consuming, limits throughput, and prevents start-to-finish automation. The NEXTFLEX® Small RNA-Seq Kit v3 allows for gel-free small RNA library preparation. This is possible thanks to the dual approach used for adapter-dimer reduction. Unprecedented reduction of adapter-dimer formation allows completely gel-free small RNA library prep when starting with ≥200 ng of total RNA. Libraries prepared with the NEXTFLEX® Small RNA-Seq kit v3 have a higher proportion of reads mapping to miRNAs (Fig. 1).
The NEXTFLEX® Small RNA-Seq kit V3 “consistently performed well with respect to enrichment of miRNA mapping reads in biofluids and tissues” and “exhibited a relatively low quantification bias”.
Low Input Small RNA Library Prep for Illumina® Sequencing
The adapter-dimer reduction technology incorporated into this kit also allows low input library preparation. Library preparation with as little as 1 ng of total RNA is possible as additional PCR cycles can be performed without adapter-dimer products dominating the final library. Fig. 2 illustrated that expression values are reproducible across different sample inputs.
Small RNA-Seq Automation Compatibility
The NEXTFLEX® Small RNA-Seq Kit V3 is designed for easy migration onto automated liquid handling platforms. Currently methods are available for the PerkinElmer Sciclone® NGS/NGSx and the Zephyr® G3 NGS workstation.
For more information contact [email protected].
Illumina® Small RNA-Seq Multiplexing
Eight barcoded primers are included in the eight reaction NEXTFLEX® Small RNA-Seq Kit v3 and forty-eight barcoded primers are included in the forty-eight reaction NEXTFLEX® Small RNA-Seq Kit v3.
Independent Analysis of Small RNA Library Prep Solutions
Six independent studies were published in 2018 and 2019 comparing small RNA sequencing data obtained using commercially available small RNA-seq library prep kits. The NEXTFLEX® Small RNA-Seq kit was recommended in every study because of the low bias and consistent results obtained using the kit.
Chu, C. P., & Nabity, M. B. (2019). Comparison of RNA isolation and library preparation methods for small RNA sequencing of canine biofluids. Veterinary Clinical Pathology. doi:10.1111/vcp.12743
Coenen-Stass, A.M.L., et al. (2018) Evaluation of methodologies for microRNA biomarker detection by next generation sequencing. RNA Biology. 15: 8. 15:8, 1133-1145. doi: 10.1080/15476286.2018.1514236.
Dard-Dascot, C., et al. (2018) Systematic comparison of small RNA library preparation protocols for next-generation sequencing. BMC Genomics 19(118), doi:10.1186/s12864-018-4491-6.
Giraldez, M. D., Spengler, R. M., Etheridge, A., Godoy, P. M., Barczak, A. J., Srinivasan, S., . . . Tewari, M. (2018). Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling. Nature Biotechnology. doi:10.1038/nbt.4183.
Wright, C., Rajpurohit, A., Burke, E. E., Williams, C., Collado-Torres, L., Kimos, M., . . . Shin, J. H. (2018). Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods. bioRxiv 445437. doi:10.1101/445437.
Yeri, A., et al. (2018) Evaluation of commercially available small RNASeq library preparation kits using low input RNA. BMC Genomics 201819:331. doi: 10.1186/s12864-018-4726-6.
- NEXTFLEX® 3’ 4N Adenylated Adapter
- NEXTFLEX® 3’ Ligation Buffer
- NEXTFLEX® 3’ Ligation Enzyme Mix
- NEXTFLEX® Adapter Depletion Solution
- NEXTFLEX® Adapter Inactivation Buffer
- NEXTFLEX® Adapter Inactivation Enzyme
- NEXTFLEX® 5’ 4N Adapter
- NEXTFLEX® 5’ Ligation Buffer
- NEXTFLEX® 5’ Ligation Enzyme Mix
- M-MuLV Reverse Transcriptase
- NEXTFLEX® RT Buffer
- NEXTFLEX® Universal Primer
- NEXTFLEX® Barcode Primer
- NEXTFLEX® Small RNA PCR Master Mix
- 6X Loading Dye
- Ready to Load Low MW Ladder
- Resuspension Buffer
- Nuclease-free Water
- microRNA Control
- NEXTFLEX® Cleanup Beads
- NEXTFLEX® Elution Buffer
REQUIRED MATERIALS NOT PROVIDED
- 1 ng – 2 μg total RNA or purified small RNA from 1-10 μg total RNA in up to 10.5 μL Nuclease-free Water
- 80% Ethanol
- 2, 10, 20, 200 and 1000 μL pipettes
- RNase-free pipette tips
- 96 well PCR Plate Non-skirted (Phenix Research®, Cat # MPS-499) or similar
- Thin-wall nuclease-free PCR tubes
- Heat block
- Magnetic Stand -96 (Thermo Fisher Scientific®, Cat # AM10027) or similar
SEQUENCES & INDICES
- Sequences of NEXTFLEX Small RNA-Seq Barcode Indexes – Excel / PDF
- Instructions for installing NEXTFLEX Barcode Indices in Illumina® Experiment Manager
- Download the Sage® Pippin Prep® Protocol
- Download the Small RNA Trimming Instructions
- Download the No Size Selection Protocol
ON-DEMAND GENOMEWEB WEBINAR
- Increasing Ligation Efficiency and Discovery of miRNAs for Small RNA NGS Sequencing Library Prep with Plant Samples
- Reduced-Bias Small RNA Library Preparation with Gel-Free or Low-Input Options
Selected Citations that Reference the Use of the NEXTFLEX Small RNA-Seq Kit V3:
- Carney, M. C., Tarasiuk, A., DiAngelo, S. L., Silveyra, P., Podany, A., Birch, L. L., … & Hicks, S. D. (2017). Metabolism-related microRNAs in maternal breast milk are influenced by premature delivery. Pediatric research, 82(2), 226.
- Chen, Y., Wang, J., Yang, S., Utturkar, S., Crodian, J., Cummings, S., & Plaut, K. (2017). Effect of high-fat diet on secreted milk transcriptome in midlactation mice. Physiological genomics, 49(12), 747-762.
- Chotewutmontri, P., Stiffler, N., Watkins, K. P., & Barkan, A. (2018). Ribosome Profiling in Maize. In Maize (pp. 165-183). Humana Press, New York, NY.
- Chu, C. P., & Nabity, M. B. (2019). Comparison of RNA isolation and library preparation methods for small RNA sequencing of canine biofluids. Veterinary Clinical Pathology. doi:10.1111/vcp.12743.
- Coenen-Stass, A.M.L., et al. (2018) Evaluation of methodologies for microRNA biomarker detection by next generation sequencing. RNA Biology. 15: 8. 15:8, 1133-1145. doi: 10.1080/15476286.2018.1514236.
- Dard-Dascot, C., et al. (2018) Systematic comparison of small RNA library preparation protocols for next-generation sequencing. BMC Genomics 19(118), doi:10.1186/s12864-018-4491-6.
- Fu, F. et al. (2018) Loss of mCHH islands in maize chromomethylase and DDM1-type nucleosome remodeler mutants. dx.doi.org/10.1101/253567.
- Garcia-Elias, A. et al. (2017) Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs. Scientific Reports. 7: 7725. doi:10.1038/s41598-017-08134-3.
- Ghasemzadeh, A., ter Haar, M. M., Shams-bakhsh, M., Pirovano, W., & Pantaleo, V. (2018). Shannon entropy to evaluate substitution rate variation among viral nucleotide positions in datasets of viral siRNAs. In Viral Metagenomics (pp. 187-195). Humana Press, New York, NY.
- Giraldez, M. D., Spengler, R. M., Etheridge, A., Godoy, P. M., Barczak, A. J., Srinivasan, S., . . . Tewari, M. (2018). Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling. Nature Biotechnology. doi:10.1038/nbt.4183
- He, R., Xie, X., Lv, L., Huang, Y., Xia, X., Chen, X., & Zhang, L. (2017). Comprehensive investigation of aberrant microRNAs expression in cells culture model of MnCl2-induced neurodegenerative disease. Biochemical and biophysical research communications, 486(2), 342-348.
- Hicks, S. D., Carney, M. C., Tarasiuk, A., DiAngelo, S. L., Birch, L. L., & Paul, I. M. (2017). Breastmilk microRNAs are stable throughout feeding and correlate with maternal weight.
- Hicks, S. D., Johnson, J., Carney, M. C., Bramley, H., Olympia, R. P., Loeffert, A. C., & Thomas, N. J. (2018). Overlapping microRNA expression in saliva and cerebrospinal fluid accurately identifies pediatric traumatic brain injury. Journal of neurotrauma, 35(1), 64-72.
- Kim, K., Yoo, D., Lee, H. S., Lee, K. J., Park, S. B., Kim, C., . . . Song, S. Y. (2019). Identification of potential biomarkers for diagnosis of pancreatic and biliary tract cancers by sequencing of serum microRNAs. BMC Medical Genomics,12(1). doi:10.1186/s12920-019-0521-8.
- Lee, E. K., Jeong, H. O., Bang, E. J., Kim, C. H., Mun, J. Y., Noh, S., & Chung, H. Y. (2018). The involvement of serum exosomal miR-500-3p and miR-770-3p in aging: modulation by calorie restriction. Oncotarget, 9(5), 5578–5587. http://doi.org/10.18632/oncotarget.23651.
- Mateescu, B., Kowal, E. J., van Balkom, B. W., Bartel, S., Bhattacharyya, S. N., Buzás, E. I., … & Driedonks, T. A. (2017). Obstacles and opportunities in the functional analysis of extracellular vesicle RNA–an ISEV position paper. Journal of extracellular vesicles, 6(1), 1286095.
- Miranda, R. G., McDermott, J. J., & Barkan, A. (2017). RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR–RNA interactions. Nucleic acids research.
- Nguyen, Q., Iritani, A., Ohkita, S., Vu, B. V., Yokoya, K., Matsubara, A., & Nakayashiki, H. (2018). A fungal Argonaute interferes with RNA interference. Nucleic acids research.
- Ong, J., Woldhuis, R. R., Boudewijn, I. M., Berg, A. V., Kluiver, J., Kok, K., . . . Brandsma, C. A. (2019). Age-related gene and miRNA expression changes in airways of healthy individuals. Scientific Reports,9(1). doi:10.1038/s41598-019-39873-0.
- Pinti, M. V., Hathaway, Q. A., Kunovac, A., Durr, A. J., Cook, C. C., Roberts, H. G., Salman, M., and Hollander, J. M. (2019) microRNA Changes in Diabetic Cardiac Mitochondria: What are they doing there? FASEB J. doi:10.1096/fasebj.2019.33.1_supplement.713.3.
- Rafael G Miranda, James J McDermott, Alice Barkan; RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR–RNA interactions, Nucleic Acids Research, Volume 46, Issue 5, 16 March 2018, Pages 2613–2623, https://doi.org/10.1093/nar/gkx1288.
- Rosenberg, A. Z., Wright, C., Fox-Talbot, K., Rajpurohit, A., Williams, C., Porter, C., . . . Halushka, M. K. (2018). XMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells. doi:10.1101/333658.
- Wright, C., Rajpurohit, A., Burke, E. E., Williams, C., Collado-Torres, L., Kimos, M., . . . Shin, J. H. (2018). Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods. bioRxiv 445437. doi:10.1101/445437.
- Yeri, A., et al. (2018) Evaluation of commercially available small RNASeq library preparation kits using low input RNA. BMC Genomics 201819:331. doi: 10.1186/s12864-018-4726-6.
Depletion of tRNA and YRNA from Plasma for Small RNA Sequencing
The NEXTFLEX® Small RNA-Seq Kit v3 is compatible with cell-free RNA, such as RNA isolated from plasma. Users who wish to deplete the abundant tRNA fragments and Y RNA fragments found in many types of cell-free RNA should use the NEXTFLEX® tRNA/YRNA Blocker (not included in kit). Please contact us at [email protected] if interested. Download instructions here.
Using the NEXTFLEX Small RNA-Seq Kit v3 for alternative RNA inputs, including RIP-Seq, CLIP-Seq, HITS-CLIP, and Ribosomal Profiling
Although NEXTFLEX Small RNA Sequencing Kits are normally used to prepare sequencing libraries representing miRNAs, siRNAs, or piRNAs, the kits can also be used to create libraries from other RNA samples, such as RNA isolated from ribosomal profiling, RIP-Seq (RNA binding protein immunoprecipitation and sequencing), CLIP-Seq (cross-linking immunoprecipitation and sequencing), and HITS-CLIP experiments. To create sequencing libraries with NEXTflex Small RNA Sequencing Kits, the RNA of interest should have a 5′ monophosphate and 3′ hydroxyl. Fortunately, these modifications can easily be added to most RNA molecules with T4 polynucleotide kinase (PNK). PNK contains both 5′ kinase and 3′ phosphatase activities, making it ideal for preparing RNA samples for library preparation with the NEXTFLEX Small RNA Seq Kit v3.
In order to prepare a library from RNA molecules that do not already contain a 5′ monophosphate and 3′ hydroxyl, the following basic strategy can be followed:
- Treat RNA with T4 PNK according to the manufacturer’s instructions. A 10 minute pre-incubation with all components except ATP may help increase the phosphatase activity, which will lead to greater yield for RNA samples that contain 3′ phosphates, such as those that have undergone chemical/heat fragmentation.
- Purify RNA using ethanol precipitation or a column-based kit, such as the Zymo® RNA Clean and Concentrator-5. Resuspend/elute the RNA in ~12 µL of water.
- Optional Check RNA size and approximate quantity by Agilent® Bioanalyzer®. Note that Bioanalyzer® estimates of concentration are often inaccurate, so this value should be treated as an estimate.
- Prepare libraries using the NEXTFLEX Small RNA-Seq Kit v3. If ≥ 1 ng of PNK-treated RNA is used for library prep, 15 cycles or fewer of PCR should be sufficient.
The default protocol enriches for final library products representing RNA molecules of ~20-30 nt. In order to retain products representing larger RNA fragments, the protocol should be modified according to the instructions in the NEXTFLEX Small RNA Seq Kit v3 No Size Selection Supplement.
- If a final size selection is desired, gel-based size selection can be performed, or the volumes used in Step H1: Gel-Free Size Selection & Cleanup can be optimized for the desired size range. Note that bead-based methods only achieve coarse size selection, so PAGE purification is recommended if precise size selection is needed.
Can the Sage® Pippin Prep® system be used for automated size selection of small RNA libraries created with the NEXTFLEX Small RNA Sequencing Kit v3?
Yes, a protocol is available for automated size selection of small RNA libraries constructed using the NEXTFLEX Small RNA Sequencing Kit v3. Download the Sage® Pippin Prep® protocol for the NEXTFLEX Small RNA-Seq Kit v3.
What is the lowest input of total RNA possible with this kit?
The lowest recommended input is 1 ng of total RNA. This amount of RNA should work well for most sample types. For samples that have high miRNA content, less total RNA may be used.
Why do I have to perform a cleanup after 3′ ligation? This step is not necessary in other kits.
Typical “tricks” to reduce formation of adapter-dimer do not work well when using adapters with randomized ends, which is the reason for the Excess 3′ Adapter Depletion step.
Is this kit compatible with both single read and paired-end sequencing?
Yes, in addition to single read sequencing this kit can also be used with paired-end sequencing.
Will the random nucleotides from the adapters be present in my sequencing reads?
Yes, the random bases will be present as the first four bases of the read and the four bases immediately before the 3′ adapter sequence.
How should the random bases be handled for alignment?
Following 3′ adapter trimming, the first and last four bases of the read should be trimmed prior to alignment. Another option is to use an aligner with a “local” mode.
What sequencing platforms is this kit compatible with?
This kit is compatible with all common Illumina® sequencing platforms including the HiSeq®, MiSeq®, and NextSeq® 500 systems.
How many sequencing cycles do small RNA libraries need?
This is dependent on your experiment and whether you’re looking at microRNAs or long non-coding RNAs. For microRNAs, we do not recommend fewer than 36 sequencing cycles. The Illumina® MiSeq® 1×50 cycle run is most commonly used for sequencing microRNAs. Most microRNAs are between 15–30 bases long, and sequencing beyond this point only sequences the adapter.
How many reads are needed per sample for small RNA sequencing?
This is also dependent on your experiment. Generally for expression profiling, 1–2M mapped reads is an accepted range. For discovery applications, you may want to increase to 2–5M reads. Contact us at [email protected] and we can help you determine the best number of reads based on your experimental design.
How are the barcoded primers incorporated into the small RNA-Seq libraries?
The NEXTFLEX Small RNA Barcodes Primers, each containing a six-base index, are incorporated into the library during the PCR amplification step. This design allows for the indexes to be read using a second read, which reduces bias.
During size selection on 6% PAGE gel, which bands should I cut out of the gel?
MicroRNAs that are between 20 – 30 nt in length will yield a band ~150 -160 bp in length, which should be cut out of the 6% PAGE gel. Do not cut out the ~130 bp band as this is adapter dimer.
What is AIR™ Ligase and why is it used in the NEXTFLEX Small RNA-Seq Kit v3?
AIR Ligase is an enhanced, truncated T4 RNA Ligase, which increases the efficiency with which small RNAs are tagged with adapter, giving greater sequence depth.
This kit recommends up to 25 cycles of PCR amplification for low-input libraries. Won’t this many PCR cycles intro bias?
No, PCR has been shown to introduce negligible bias into small RNA libraries. See publications by Jayaprakash, et al., Hafner, et al. and our whitepaper for more information.
Jayaprakash, A.D., et al., Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res, 2011. 39(21): p. e141.
Hafner, M., et al., RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA, 2011. 17(9): p. 1697-712.