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Bioo Scientific - Reduced Bias Small RNA Library Prep with Gel-Free or Low-Input Options

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microRNAs (miRNAs) may provide useful markers for the development of disease diagnostic and prognostic assays. NGS brings sensitivity, specificity, and the ability to maximize data acquisition and minimize costs of miRNA sequencing by using multiplex strategies to allow many samples to be sequenced simultaneously with small RNA analysis. However, small RNA sequencing has typically suffered from three major drawbacks: severe bias, such that sequencing data does not reflect original miRNA abundances, the need to gel purify final libraries, and lack of low-input protocols. The NEXTflex™ Small RNA-Seq Kit v3 addresses these drawbacks by using two strategies: randomized adapters to reduce ligation-associated bias, and a dual approach to adapter-dimer reduction, thereby allowing gel-free or low-input small RNA library preparation.

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Bioo Scientific - Reduced Bias Small RNA Library Prep with Gel-Free or Low-Input Options

  1. 1. Reduced Bias Small RNA Library Prep with Gel-Free or Low-Input Options HERD TOGETHER. ®
  2. 2. Small RNA sequencing is inherently saddled with three distinct PROBLEMS!
  3. 3. Substantial bias introduced during ligation steps Need to gel purify final library Lack of low-input protocols PROBLEM # 1 PROBLEM # 2 PROBLEM # 3
  4. 4. PROBLEM # 1 Research shows extensive bias is typically seen in small RNA sequencing Baran-Gale, J., Kurtz, L. C., Erdos, Sison, M. C., Young, A., Fannin, E. E., Chines, P. S. and Sethupathy, P. (2015) Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade capture by current methods. Frontiers in Genetics. doi: 10.3389/fgene.2015.00352.
  5. 5. What causes this bias?
  6. 6. Research shows these inconsistencies are primarily caused by bias introduced during the ligation step of library prep Jayaprakash, A. D., Jabado O., Brown, B. D. and Sachidanandam, R. (Sept 2, 2011), Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing Nuc Acid Res, 1–12. doi:10.1093/nar/gkr693 Zhang, et al. (2013) High-efficiency cloning enables accurate quantification of miRNA expression by deep sequencing. Genome Biology 14 R109. Sun, G. (2011) A bias-reducing strategy in profiling small RNAs using Solexa. RNA. 17: 2256-2262 Sorefan, K. et al. (2012) Reducing sequencing bias of small RNAs. Silence. doi:10.1186/1758-907X-3-4
  7. 7. What is the SOLUTION?
  8. 8. Published research shows randomized adapters reduce ligation bias Jayaprakash, A. D., Jabado O., Brown, B. D. and Sachidanandam, R. (Sept 2, 2011), Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing Nuc Acid Res, 1–12. doi:10.1093/nar/gkr693.
  9. 9. Bioo Scientific makes the only small RNA sequencing kits for Illumina platforms that use randomized adapters to reduce ligase bias
  10. 10. The NEXTflex Small RNA-Seq Kit v3 shows more equal coverage of an equimolar pool of 24 miRNAs (miRNA calibrator), demonstrating more accurate representation of the original sample
  11. 11. Figure 1. Sequencing results from small RNA libraries created in triplicate from 1 ng of miRNA Calibrator, an equimolar mixture of 24 miRNAs. Values farther from 1 indicate more bias. 100 10 1 0.1 0.01 0.001 Observed/Expected miRNA Calibrator hsa-miR-92b-5p hsa-miR-324-3p dme-miR-6-3p dme-miR-4-3p has-miR-134 has-miR-23a-5p hsa-miR-133a hsa-miR-127-5p hsa-miR-24-3p hsa-let-7e-3p hsa-miR-195-3p hsa-miR-92a-3p hsa-miR-34c-3p hsa-miR-30c-1-3p hsa-miR-320a hsa-miR-218-1-3p hsa-miR-34a-5p hsa-miR-106b-5p hsa-miR-141-3p hsa-let-7c hsa-miR-15a-5p hsa-miR-21-5p hsa-miR-29b-3p hsa-miR-190a CV NEXTflex = 1.09 Illumina = 2.35 NEB = 2.31 NEXTflex Illumina NEB
  12. 12. The NEXTflex Small RNA-Seq Kit v3 shows more even coverage with the Miltenyi miRXplore Universal References, an equimolar mixture of 963 miRNAs
  13. 13. Figure 2. Sequencing results from small RNA libraries created in triplicate from 1 ng of Miltenyi miRXplore Universal Reference, an equimolar mixture of 963 miRNAs. 1000 900 800 700 600 500 400 300 200 100 0 0 miRNAsdetected miRNAs detected Threshold (reads/100K) NEXTflex Illumina NEB 10 20 30 40 50 60 70 80 90 100 CV NEXTflex = 1.14 Illumina = 3.78 NEB = 1.67
  14. 14. The use of randomized adapters in the NEXTflex Small RNA-Seq Kit v3 greatly improves accuracy of data by reducing bias in small RNA library prep CONCLUSION # 1
  15. 15. The NEXTflex Small RNA-Seq Kit v3 allows detection of more miRNAs in total RNA samples
  16. 16. Figure 3. Small RNA libraries were created in duplicate from human brain total RNA and sequenced on an Illumina MiSeq. The indicated number of reads was sampled from each library and the average number of miRNA groups with ≥20 reads determined. The inset shows the number of reads required to detect 100 miRNA groups at a threshold of ≥20 reads. Sequencing depth versus miRNAs detected mirRNAgroupswith≥20reads Reads sampled 180 160 140 120 100 80 60 40 20 0 0 50000 100000 150000 200000 NEXTflex - 100 ng NEXTflex - 10 ng NEB - 100 ng Illumina - 100 ng
  17. 17. The NEXTflex Small RNA-Seq Kit v3 detects the same number of miRNAs with fewer reads
  18. 18. Figure 4. 3 - 4.5x fewer reads are necessary to detect 100 miRNAs with the NEXTflex Small RNA Seq-Kit v3. Reads necessary to detect 100 miRNAs 200000 150000 100000 50000 0 NEXTflex - 100 ng NEXTflex - 10 ng NEB - 100 ng Illumina - 100 ng
  19. 19. Reduced bias small RNA library prep using the NEXTflex Small RNA-Seq Kit v3 allows detection of more small RNAs at lower sequencing depth CONCLUSION # 2
  20. 20. PROBLEM # 2 Small RNA library preparation has historically required PAGE gel purification due to the presence of adapter-dimer products
  21. 21. PAGE purification is tedious & time consuming
  22. 22. PAGE purification limits throughput & prevents start-to-finish automation
  23. 23. Elimination of adapter-dimer products allows gel-free purification of small RNA libraries
  24. 24. The NEXTflex Small RNA-Seq Kit v3 uses a dual approach to substantially reduce adapter-dimer formation, allowing gel-free purification of final libraries
  25. 25. Gel-free libraries prepared with the NEXTflex Small RNA-Seq Kit v3 have a higher proportion of reads mapping to miRNAs
  26. 26. Figure 5. Percent of total reads aligned to miRBase in gel-free libraries created from 100 ng human brain total RNA input. 0 10 20 30 40 50 60 70 Bioo-100 ng Bioo- 10 ng %miRBasealigned Overall alignment rate% miRBase alignment NEXTflex NEB 70 60 50 40 30 20 10 0
  27. 27. Enhanced reduction of adapter-dimer formation allows gel-free library preparation CONCLUSION # 3
  28. 28. PROBLEM # 3 Lack of low-input protocols
  29. 29. Substantial reduction of adapter-dimers also allows low input library preparation
  30. 30. Libraries can be prepared from as little as 1 ng total RNA
  31. 31. Low input libraries are achieved by using up to 25 cycles of PCR
  32. 32. PCR bias adds negligible bias to small RNA libraries
  33. 33. Correlation of miRNA expression demonstrates negligible bias added by additional PCR cycles PCR Cycles 12 16 20 24 28 12 1 0.9999 0.9994 0.9966 0.9807 16 1 0.9996 0.9970 0.9813 20 1 0.9975 0.9824 24 1 0.9816 28 1 Table 1. Correlation of miRNA abundance in libraries created using serial 10x dilutions of cDNA and the indicated number of PCR cycles. The Pearson correlation coefficients calculated from the Log10(reads) values of miRNAs with ≥ 10 reads in all samples are shown
  34. 34. Published data shows that additional PCR cycles add negligible bias to small RNA libraries 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.
  35. 35. Expression values are reproducible across different sample inputs Figure 6. Correlation of miRNA expression between samples created with 100 ng and 10 ng of human brain total RNA with the NEXTflex Small RNA-Seq kit v3. The Pearson correlation coefficient is shown. 1 1.5 2 2.5 3 3.5 4 4.5 5 1 1.5 2 2.5 3 3.5 4 4.5 5 Log10(reads)100ng Log10(reads) 10 ng 100 ng vs 10 ng100 ng vs 10 ngLog10(reads)100ng Log10(reads) 100 ng 5 4.5 4 3.5 3 2.5 2 1.5 1 1 1.5 2 2.5 3 3.5 4 4.5 5 R = 0.964
  36. 36. Gel purification of low-input small RNA libraries is still necessary
  37. 37. Enhanced reduction of adapter-dimer formation allows library preparation from as little as 1 ng of total RNA CONCLUSION # 4
  38. 38. The NEXTflex Small RNA-Seq Kit v3 is the only commercially available kit that includes randomized adapters to reduce bias and allows gel-free or low-input library prep
  39. 39. NEXTflex™ Small RNA-Seq Kit v3 (Illumina® Compatible) • 48 unique barcodes for multiplexing included with 48 reaction kit • 8 reaction kit includes barcodes that allow low-level multiplexing
  40. 40. Learn more about the NEXTflex™ Small RNA-Seq Kit v3 to obtain more accurate results from small RNA sequencing NEXTflex™ Small RNA-Seq Kit v3 (Illumina Compatible) Catalog #: 5132-05 (8 reactions) GEL-FREE & LOW INPUT OPTIONS ©Bioo Scientific Corporation · 2015 V15.11 ® ® nextgen@biooscientific.com THE NGS EXPERTS™ Reduced-Bias Small RNA Library Preparation with Gel-Free or Low-Input Options INTRODUCTION Changes in microRNA (miRNA) expression have been shown to be associated with a variety of normal physiological processes, as well as diseases including cancer. Studies have already shown that miRNAs may provide useful markers for the development of disease - Bias is introduced into small RNA libraries during the ligation steps. Small RNA library preparation involves ligating adapters directly onto the 3’ and 5’ ends of the RNA molecule in two separate steps, and each of these ligation steps has been shown to introduce severe bias into library preparation. Recent studies have shown that inserting random bases at the ends of the adapters greatly reduces bias in comparison to using non-randomized adapters [1-4]. - ration. In typical DNA or RNA library prep, insert-containing molecules are at least 100 bp larger than adapter-dimer molecules, and thus can be removed using SPRI magnetic beads. However, since insert-containing molecules are only ~20 bp larger than adapter-dimer size selection greatly limits both throughput and automation potential of small RNA library preparation, as only a limited number of libraries can be run on a single gel and it is a labor-intensive process that is not amenable to automation. unique in that additional PCR cycles result in negligible bias; thus it should theoretically be possible to create low-input small RNA products and leading to sequencing data where very few of the reads are useful. A number of methods have been developed to reduce such an extent that gel-free or low-input small RNA library preparation is possible. method that utilizes randomized adapters for greatly reduced bias and features gel-free or low-input protocols, which are possible due . ® DOWNLOAD MANUAL PURCHASE KIT DOWNLOAD WHITEPAPER
  41. 41. If you have any questions email us at nextgen@biooscientific.com or visit our website at BiooScientific.com/smallRNA

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