High Resolution LabChip XT Fractionation of Illumina Compatible Small RNA Libraries using the DNA 300 Assay Kit

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1 application Note Next Generation Sequencing Authors: Lucy Sun Danh Tran Donna Fong Ryan Swenerton Elaine Wong-Ho Rajendra Singh Josh Molho Caliper Life Sciences a PerkinElmer Company Alameda, California US High Resolution LabChip XT Fractionation of Illumina Compatible Small RNA Libraries using the DNA 300 Assay Kit Introduction MicroRNAs (mirna) are short, singlestranded small RNA that control posttranscriptional gene expression, and play critical roles in biological processes. There is increasing evidence that mirnas have important functions in human diseases including cancer. 1 Currently, mirnas are being extensively studied and profiled by next-generation sequencing. Generating accurate sequencing data requires starting with high quality small RNA libraries. Fractionation of nucleic acid is a time consuming process, but it is a critical part of library construction. Conventional gel-based methods are notoriously inefficient and introduce variability that can negatively impact data quality. The LabChip XT Nucleic Acid Fractionation System performs automated and accurate nucleic acid sizing, processes up to four samples in less than 60 minutes, and delivers fractionated samples in a sequencing compatible buffer. The fragment size distribution of a small RNA library is relatively narrow, and accurate sizing is particularly important for removing artifacts such as adapterdimers introduced during the library construction process. Separating fragments containing the library from no-insert fragments significantly improves the amount of data obtained from the sequencing reaction. The XT DNA 300 Assay provides accurate sizing and high resolution for capturing Illumina compatible small RNA libraries (NEBNext and TruSeq ), thereby minimizing the amount of adapter-dimers.

2 Total RNA Adapter ligation and reverse LabChip XT Sequencing transcription, enrichment, size fractionation and purification A B C D Figure 1. Work-flow of small RNA analysis: A) Extraction of total RNA; B) Library construction by adapter ligation, reverse transcription, enrichment, and library purification; C) Size selection using LabChip XT fractionation system; D) Sequencing. Material and Methods The small RNA library input material was generated from Human Brain Total RNA (Ambion, 6 µg/reaction) using the NEBNext Small RNA Sample Prep Set 1. From each reaction, approximately 700 ng (quantitated by HT DNA High Sensitivity Assay Kit) of library was generated, and the constructed library was purified with the QIAGEN QIAquick PCR Purification Kit before fractionation using the LabChip XT and XT DNA 300 assay. Input library ranging from ng has been tested. For details on extraction setups, see the section on Optimal Software Settings. Fractionated library was quantitated by the HT DNA High Sensitivity Assay Kit on the LabChip GX system. Sample preparation followed the Limited Sample Work-flow, fractionated samples were diluted 5 fold in water, and both the DNA Ladder and the 1x Ladder were diluted in TE. Further analysis of the fractionated library included both end-point PCR using the Phusion High-Fidelity PCR Master Mix (NEB), and Real-Time PCR (StepOne, Applied Biosystems) using the KAPA SYBR FAST Universal 2X qpcr Master Mix and DNA Quantification Standards and Primer Premix Kit/ Illumina GA (Kapa Biosystems) pg of the fractionated library was used as template for endpoint PCR. The amplified products were analyzed by the HT DNA High Sensitivity Assay after 3, 5 and 7 cycles of PCR. Template for the Real-Time PCR was fractionated library diluted at 1:1000 and 1:8000. Since the fractionated library is near 100 bp, and DNA used in the standard curve is 452 bp, final quantitation of the library was adjusted accordingly. Fractionated small RNA library at a starting concentration of 10 nm was sequenced on the Illumina HiSeq 2000 flow cell v3 using SBS sequencing kit-hs, and service was provided by the UCSF Center for Advanced Technology. Results For LabChip XT fractionation, a total input material of 130 ng library was loaded onto a chip, and an electropherogram of the extraction is shown in Figure 2A. The Skip Extraction feature of the software was used to identify and skip the adapter-dimer peak (Skip Peak in grey, 74 bp) and then to extract the subsequent library peak (Extract Peak in red, 92 bp). Both the total input library (blue) and the XT fractionated library (red) were analyzed on the LabChip GX system using the HT DNA High Sensitivity Assay to confirm purity (Figure 2B). Results show the XT fractionation delivers high quality small RNA library for sequencing with most of the adapter-dimers removed. Using an input of 300 ng library, quantitation by HT DNA High Sensitivity Assay indicates the yield of fractionated library is 24 ng at 18 nm, which is a sufficient concentration for sequencing. A. LabChip XT fractionation B. LabChip GX DNA high sense assay Figure 2. LabChip XT fractionation and analysis. 2

3 To further confirm purity of the fractionated small RNA library, it was amplified using end-point PCR, and product was analyzed by the HT DNA High Sensitivity Assay (Figure 3A). The electropherogram shows an overlay of the profiles including the original fractionated small RNA library, and the diluted library amplified after 3, 5, and 7 cycles. The PCR amplified product indicates the fractionated small RNA library is clean without contamination of adapter-dimers. The fractionated small RNA library was quantitated by Real-Time PCR and results are shown in Figure 3B - D. An amplification plot of the DNA quantification standards (grey), and the fractionated small RNA library at 1:1000 dilution (green) and 1:8000 dilution (orange) is shown in Figure 3B. Base on the amplification plot, a DNA concentration standard curve is generated, and data points for the fractionated small RNA library at 1:1000 dilution (green) and 1:8000 dilution (orange) are indicated (Figure 3C). The CT values calculated from the standard curve are tabulated in Figure 3D. For an input of 300 ng library, Real-time PCR quantitation of the fractionated small RNA library indicates the average concentration is 21 nm, which is consistent with results from the HT DNA High Sensitivity assay. Results from Illumina sequencing of the small RNA library indicate the total number of reads passing filter is 154,231,495. Analysis shows that greater than 99% of the total reads contain inserts, and only less than 0.5% of the reads is adapter-dimer sequence (Table 1). In conclusion, both the post XT fractionation analysis and sequencing data indicate the small RNA library is clean with minimal amount of the adapter-dimer. Table 1. Summary of Sequencing Data from Small RNA Library. Small RNA Library Reading Passing Filters Pass Filter Rate % Readswith Inserts 1 Cluster Density Human Brain 154,231, % % 619 ±178 K/mm 2 1 Calculated by filtering reads that started directly with the first base of the opposite end adapter sequence, may contain no or one single base insert before the adapter sequence starts assuming that the adapter sequence contain no errors. A. End-point PCR C. Real-time PCR standard curve D. Real-time PCR library quantitation B. Real-Time PCR Amplification Plot Figure 3. Fractionated small RNA library QC and quantitation. 3

4 Optimal Software Settings For most XT fractionation, a single step extraction setting is sufficient to obtain DNA with the correct size. However, for small RNA library extraction, it is necessary to extract material that is very close in size to smaller unwanted material. The Skip Extraction mode allows the user to identify and exclude the peak of smaller unwanted material (containing no insert) from the adjacent peak containing larger fragments of interest. The software will automatically identify the unwanted peak within the Search Region, and prevent the next step from triggering through the size range designated by the Collection Width. The peak identified in the Skip Extraction step will be marked in grey on the electropherogram. The optimal software settings for small RNA library fractionation have been summarized in Table 2. Figure 4. XT Run File Editor. We recommend purifying the small RNA library with QUIAGEN MinElute PCR Purification Kit prior to extraction, and should be eluted using TE or EB, not water. The software settings are selected in the XT Run File Editor as shown in Figure 4. For libraries with minimal contaminating primers, in Step 1, choose Skip Extraction for Operation, define Search Region for bp as shown in Figure 5A for Library I (NEBNext ). For samples that contain large primers due to barcode sequences, the search region might need to be adjusted to bp in order to skip the correct peak as shown in Figure 5A for Library II (TruSeq ). Choose Peak max under Trigger Mode and set Threshold for 5-50 RFU; we recommend 25 RFU for sample input of ng small RNA Library I ( ng for Library II), lower thresholds may mistake baseline noise as a peak, higher thresholds may miss a peak (Figure 5B). If the sample input is under 30 ng for Library I (50 for Library II), use a Threshold of 10 RFU. Set Collection Width for bp, depending on the width of the adapter-dimer peak. For most small RNA libraries, skip Collection Width of 25 bp is sufficient, increasing the skip Collection Width for Step 1 will delay the start of Step 2, and should reduce carryover of the unwanted peak (Figure 5C). Table 2. Summary of XT software settings. Operation Trigger Mode Threshold Start End Width Step 1 Skip Extraction Peak max 25 RFU 50 bp (100 bp) 200 bp bp Step 2 Extract and Pause Peak start 50 RFU 50 bp (100 bp) 200 bp ±5-10% 4

5 A. Search Region for Steps 1 and 2 B. Step 1 Skip Extraction Threshold Sample Input Skip Extraction Threshold < 30 ng Library I (< 50 ng Library II): 10 RFU ng Library I ( ng Library II): 25 RFU C. Step 1 Skip Extraction Collection Width For Step 2, choose Extract and Pause for Operation, define Search Region for bp (set the same Search Region as Step 1), choose Peak Start under Trigger Mode and set the Threshold for RFU (we recommend 50 RFU for most libraries, higher thresholds may miss a peak), and set Collection Width for 5-10% (depending on the width of the library peak). For libraries with larger adapters, 10% Collection Width seems to work better (Figure 5D). Using Peak Start for Step 2 minimizes the amount of possible carryover of the unwanted peak. Increasing width for Step 2 will increase the extraction time (but keep the same start) and may increase recovery. D. Step 2 Extract and Pause Collection Width Figure 5. XT extraction settings. 5

6 Conclusion The LabChip XT fractionation system performs fast, automated nucleic acid fractionation accurately and reproducibly. It is easy to use and improves laboratory efficiency by replacing the time consuming traditional gel-based size selection. The XT DNA 300 Assay provides a solution for accurate and reliable fractionation of high quality Illumina compatible small RNA libraries with a minimal amount of adapter-dimer. The optimal software settings are compatible with small RNA libraries prepared by commercially available protocols. Furthermore, fractionated library is delivered in a sequencing compatible buffer. Reference 1. Jiang, Q. et. al. Nucleic Acids Research (2009) mir2disease: a manually curated database for microrna deregulation in human disease p D98 - D104. PerkinElmer, Inc. 940 Winter Street Waltham, MA USA P: (800) or (+1) For a complete listing of our global offices, visit Copyright 2012, PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners _01 Apr 2012 Printed in USA