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Mungbean Yellow Mosaic India Virus (MYMIV)-infection, Small RNA Library Construction and Deep Sequencing for MicroRNA Identification in Vigna mungo
黑吉豆中的绿豆黄化花叶印度病毒(MYMIV)感染、sRNA库建立和鉴定microRNA的深度测序

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Abstract

This protocol describes small RNA library preparation from Vigna mungo total RNA followed by deep sequencing and analysis for microRNA identification.​

Keywords: MYMIV(MYMIV), Vigna mungo(黑吉豆), MicroRNA(microRNA), Deep sequencing(深度测序), Regards(当做)

Materials and Reagents

  1. Filter papers
  2. Plastic pots
  3. Young Vigna mungo plants
  4. Whiteflies (Bemisia tabaci)
  5. HgCl2
  6. TruSeq small RNA Library Preparation Kit (Illumina, catalog number: RS-200-0012 )
  7. Trizol reagent (Invitrogen, USA)
  8. Isopropanol
  9. Acrylamide/bisacrylamide (Merck Millipore, catalog number: 623100281001730 )
  10. T4 RNA ligase 2, truncated (New England Biolabs, catalog number: M0242 )
  11. Tris-HCl pH 7.5
  12. MgCl2
  13. DTT
  14. ATP
  15. PEG8000
  16. SuperScript® II reverse transcriptase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18064014 )
  17. TEMED (Merck Millipore, catalog number: 623171280051730 )
  18. Ammonium persulphate (Merck Millipore, catalog number: 623171100101730 )
  19. 10x TBE buffer (Thermo Fisher Scientific, AmbionTM, catalog number: AM9865 )
  20. UltraPureTM glycogen (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10814-010 )
  21. 3 M sodium acetate (Sigma-Aldrich, catalog number: S7899-100ml )
  22. Absolute ethanol (EMD Millipore, catalog number: 100983 )
  23. Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 )
  24. High Sensitivity DNA Kit (Agilent Technologies, catalog number: 5067-4626 )
  25. T4 RNA Ligase 2 buffer (see Recipes)

Equipment

  1. Electrophoresis apparatus (Bio-Rad Laboratories, model: Mini-Sub® GT Cell )
  2. Bioanalyzer (Agilent Technologies, model: Agilent 2100 )
  3. Thermocycler, DNA engine cycler (Bio-Rad Laboratories, model: PTC-0200G )
  4. Qubit 3.0 fluorometer (Life Technologies)
  5. Illumina genome analyzer IIx (Illumina)

Software

  1. Cutadapt-0.9.3
  2. Bowtie-0.12.7
  3. SeqQCv2.1
  4. BLAST (Rfam and miRBase)

Procedure

  1. Plant material and growth conditions
    1. Use young V. mungo plants for this experiment. Surface sterilize mature seeds (0.1% HgCl2 for 10 min) and rinse twice in double-distilled water and keep in moistened filter papers for germination at 28 ± 2 °C, 16/8 h light/dark photoperiod and 70% relative humidity for 1 day. Perform three replicates of this experiment.
    2. Sown pre-germinated seeds in plastic pots and grow under controlled greenhouse conditions (temperature 26 ± 2 °C, relative humidity 70% and photoperiod 16 h day and 8 h night) in Soilrite soil mix (mixture of exfoliated vermiculite, Irish peat moss and perlite in the ratio 1/3:1/3:1/3).
    3. MYMIV-stress was given to plants with similar growth when their first trifoliate leaf expanded completely (approx. 21 days after sprouting).


      Figure1. Yellow mosaic symptoms on Vigna leaves

  2. MYMIV-stress treatments
    1. Rear white fly (Bemisia tabaci) populations on susceptible black gram plants grown in insect proof cages maintained in the insectory facility at the experimental farm, Madhyamgram, Bose Institute.
    2. Capture about 25-30 adult white flies in transparent glass trappers and allowed for a 24 h acquisition access period (AAP) on symptomatic leaves of naturally infected black gram plants (Figure 1).
    3. Give a 24 h inoculation access period (IAP) in the glass trappers with viruliferous whiteflies on the leaves of healthy plants, thereby transmitting the virus particles from the viruliferous flies to the plant (Figure 2).


      Figure 2. Trapper method for MYMIV inoculation

    4. Confirm the infection by PCR amplification of the MYMIV coat protein (575 bp fragment; Gen-Bank Accession number: HQ221570) using the primers MYMIV- coat protein (CP) gene (CP-F: 5’-GAA ACCTCGGTTTTACCG ACTGTATA G-3’ and CP-R: 5’-TTGCATA CACAGGATTTG AGGCATGAG-3’).

  3. Small RNA library preparation
    1. Construct small RNA libraries for sequencing according to the Illumina TruSeq small RNA library protocol outlined in 'TruSeq small RNA sample preparation guide' (Part # 15004197; Rev. E; February 2013) (Figure 3).
    2. Manually isolate10 µg of total RNA (with a RIN value of 8 and above) using Trizol reagent and concentrate by 2 volumes chilled isopropanol, then resolve in a 15% denaturing polyacrylamide gel, and then excise small RNA fragments in the range of 18-0 nt from the gel and purify. Ligate 3’ and 5’ adaptors to the small RNAs using T4 RNA Ligase 2 in a buffer (see Recipe 1) by incubating at 25 °C for 1 h.
    3. Reverse transcribe the ligated products with Superscript II reverse transcriptase by priming with adapter specific primers in 15 µl reaction mixture containing 0.15 µl dNTP mix (100 mM), 1 µl reverse transcriptase (50 U/µl), 10x buffer (1.5 µl), RNase inhibitor (0.2 µl), water (4.2 µl), 3 µl RT primer and 5 µl RNA. Incubated the reaction mixture at 42 °C for 1 h followed by a heat inactivation of 5 min at 85 °C.
    4. Enriched the cDNA by a low cycle PCR amplification of 15 cycles (initial denaturation of 30 sec at 98 °C; 15 cycles of 98 °C for 10 sec, 60 °C for 30 sec, and 72 °C for 15 sec; followed by a final extension at 72 °C for 10 min) to generate ample template for deep sequencing and clean using polyacrylamide gel followed by overnight gel elution and salt precipitation using glycogen, 3 M sodium acetate and absolute ethanol. Re-suspended the precipitate in nuclease free water.
    5. Quantify the prepared library using Qubit fluorometer (50 ng/µl), and validate for quality by running an aliquot on high sensitivity DNA kits.


      Figure3. Flow chart for TruSeq small RNA sample preparation

    6. Analyse small RNA using softwares/programmes like cutadapt-0.9.3 (Martin, 2011), bowtie-0.12.7 (Langmead and Salzberg, 2012), SeqQCv2.1 (DeLuca et al., 2012) and BLAST (Altschul et al., 1990). Steps of analysis are as under:
      1. Sequencing samples read files
        Generate fastq files from sequencing on GAIIx and use for downstream analysis.
      2. Sequence reads quality control (QC) reports
        Perform QC for all the generated sequence reads from different samples in different lanes (Mugasimangalam et al., manuscript under preparation). Consider QC report for all the sequencing reads on the basis of percentage of high quality reads which has > 70% of bases in a read with > 20 phred score. Based on the analysis as per the QC (HQ reads > 90%) report process the sequenced reads further.
      3. Trimming reads
        Trimming is done using cutadapt-0.9.3 to remove ligated adapter sequences from the original small RNA sequences. Cutadapt tool removes user specified sequences from the fastq files (Martin, 2011).
      4. Generating unique reads and counting
        Identify unique reads by writing the custom scripts and generate the read counts. Briefly below mentioned steps were carried out:
        Remove the reads which were less than or equal to 15 bases in length. Consider reads whose lengths were in the range of 16 to 36 bases. Identify unique sequences and remove duplicate sequences. Generate read count for each unique reads by identifying how many times it is present in the sequence data.
      5. Annotations of SnoRNA, SnRNA, rRNA, tRNA
        Align the sequences against Glycine max genome using bowtie-0.12.7. Bowtie supports end-to-end alignment of fastq reads to the reference fasta file (Langmead et al., 2009). End-to-end alignment considers only those reads which align completely with upto 3 mismatches to the reference fasta file. Check the reads aligning with upto 3 mismatches by BLASTn (Altschul et al., 1990) against Rfam database (using default parameters) to identify other small RNA in the sequenced data (Meyers et al., 2008).
      6. miRNA annotation
        Check the unique sequences by BLASTn (with an E-value cutoff of 10) against miRbase database to identify known miRNAs. Blast identifies similar sequences based on the sequence homology between the reference and query (unique reads).

Recipes

  1. T4 RNA Ligase 2 buffer
    31 mM Tris-HCl pH 7.5
    6.3 mM MgCl2
    0.63 mM DTT
    0.2 mM ATP
    5.2% PEG8000

Acknowledgments

The original version of this protocol was described in Paul et al. (2014). This work was supported by the Council of Scientific and Industrial Research, New-Delhi, India for the Emeritus Scientist’s Project [Sanction No. 21 (0884)/12/EMR-II].

References

  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215(3): 403-410.
  2. DeLuca, D. S., Levin, J. Z., Sivachenko, A., Fennell, T., Nazaire, M. D., Williams, C., Reich, M., Winckler, W. and Getz, G. (2012). RNA-SeQC: RNA-seq metrics for quality control and process optimization. Bioinformatics 28(11): 1530-1532.
  3. Langmead, B., Trapnell, C., Pop, M. and Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3): R25.
  4. Langmead, B. and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4): 357-359.
  5. Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMB J 17(1): pp. 10-12.
  6. Meyers, B. C., Axtell, M. J., Bartel, B., Bartel, D. P., Baulcombe, D., Bowman, J. L., Cao, X., Carrington, J. C., Chen, X., Green, P. J., Griffiths-Jones, S., Jacobsen, S. E., Mallory, A. C., Martienssen, R. A., Poethig, R. S., Qi, Y., Vaucheret, H., Voinnet, O., Watanabe, Y., Weigel, D. and Zhu, J. K. (2008). Criteria for annotation of plant MicroRNAs. Plant Cell 20(12): 3186-3190.
  7. Mugasimangalam, R., Mohan, K., Jayakumar, V., Ashick, M. M., Kapila, G. and Niranjan, V. SeqQC: rapid quality control of next generation sequence data (manuscript under preparation).
  8. Paul, S., Kundu, A. and Pal, A. (2014). Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing. J Integr Plant Biol 56(1): 15-23.
  9. TruSeqsmall RNA sample preparation guide. Illumina Inc.

简介

这个协议描述从猕猴真菌总RNA的小RNA文库制备,然后深度测序和分析microRNA识别。

关键字:MYMIV, 黑吉豆, microRNA, 深度测序, 当做

材料和试剂

  1. 过滤纸
  2. 塑料罐
  3. 年轻 Vigna mungo 植物
  4. 白蛾( Bemisia tabaci )
  5. HgCl <2>
  6. TruSeq小RNA文库制备试剂盒(Illumina,目录号:RS-200-0012)
  7. Trizol试剂(Invitrogen,USA)
  8. 异丙醇
  9. 丙烯酰胺/双丙烯酰胺(Merck Millipore,目录号:623100281001730)
  10. T4 RNA连接酶2,截短(New England Biolabs,目录号:M0242)
  11. Tris-HCl pH 7.5
  12. MgCl 2
  13. DTT
  14. ATP
  15. PEG8000
  16. SuperScript II逆转录酶(Thermo Fisher Scientific,Invitrogen TM ,目录号:18064014)
  17. TEMED(Merck Millipore,目录号:623171280051730)
  18. 过硫酸铵(默克密理博公司,目录号:623171100101730)
  19. 10x TBE缓冲液(Thermo Fisher Scientific,Ambion TM ,目录号:AM9865)
  20. UltraPure TM糖原(Thermo Fisher Scientific,Invitrogen TM ,目录号:10814-010)
  21. 3 M乙酸钠(Sigma-Aldrich,目录号:S7899-100ml)
  22. 无水乙醇(EMD Millipore,目录号:100983)
  23. dsDNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q32854)
  24. 高灵敏度DNA试剂盒(Agilent Technologies,目录号:5067-4626)
  25. T4 RNA连接酶2缓冲液(参见配方)

设备

  1. 电泳装置(Bio-Rad Laboratories,型号:Mini-Sub GT Cell)
  2. 生物分析仪(Agilent Technologies,型号:Agilent 2100)
  3. 热循环仪,DNA引物循环仪(Bio-Rad Laboratories,型号:PTC-0200G)
  4. Qubit 3.0荧光计(Life Technologies)
  5. Illumina基因组分析仪IIx(Illumina)

软件

  1. Cutadapt-0.9.3
  2. Bowtie-0.12.7
  3. SeqQCv2.1
  4. BLAST(Rfam和miRBase)

程序

  1. 植物材料和生长条件
    1. 使用young V。 mungo 植物。表面消毒成熟种子(0.1%HgCl 2 10分钟),并在双蒸水中漂洗两次并保持在润湿的滤纸中以在28±2℃,16/8h光/暗处发芽光周期和70%相对湿度下培养1天。执行此实验的三次重复。
    2. 在塑料盆中播种预发芽的种子,并且在Soilrite土壤混合物(剥落的蛭石,爱尔兰泥炭苔和混合物)中在受控的温室条件(温度26±2℃,相对湿度70%,光周期16小时和8小时夜)珍珠岩以1/3:1/3:1/3的比例)。
    3. 当它们的第一片三叶叶完全膨胀(发芽后约21天)时,给予具有类似生长的植物的MYMIV胁迫。


      图1。
  2. MYMIV应激治疗
    1. 在保持在实验农场,Madhyamgram,Bose Institute的保育设施中的昆虫证明笼中生长的易感黑色植物上的后白蝇( Bemisia tabaci )。
    2. 在透明玻璃捕捉器中捕获约25-30个成年白蝇,并且在自然感染的黑色植物的有症状的叶子上允许24小时获取进入期(AAP)(图1)。
    3. 在健康植物叶片上的有毒粉虱的玻璃捕捉器中给予24小时接种进入期(IAP),从而将病毒颗粒从有毒的苍蝇传播到植物(图2)。


      图2. MYMIV接种的Trapper方法

    4. 使用引物MYMIV-外壳蛋白(CP)基因(CP-F:5'-GAA ACCTCGGTTTTACCGACTGTATA G-3'),通过PCR扩增MYMIV外壳蛋白(575bp片段; Gen-Bank登录号:HQ221570)和CP-R:5'-TTGCATA CACAGGATTTG AGGCATGAG-3')。

  3. 小RNA文库制备
    1. 根据'中概述的Illumina TruSeq小RNA文库操作程序构建小RNA文库以进行测序 TruSeq small RNA sample preparation guide '(Part#15004197; Rev. E; February 2013)(图3)。
    2. 使用Trizol试剂手动分离10μg总RNA(RIN值为8及以上),并用2倍体积的冷冻异丙醇浓缩,然后在15%变性聚丙烯酰胺凝胶中分离,然后切除18-0的范围内的小RNA片段并从凝胶中纯化。使用T4 RNA连接酶2在缓冲液(参见配方1)中通过在25℃孵育1小时将3'和5'接头连接到小RNA。
    3. 通过用15μl含有0.15μldNTP混合物(100mM),1μl逆转录酶(50U /μl),10×缓冲液(1.5μl)的反应混合物中的衔接子特异性引物引发用Superscript II逆转录酶逆转录连接的产物, ,RNA酶抑制剂(0.2μl),水(4.2μl),3μlRT引物和5μlRNA。将反应混合物在42℃下孵育1小时,随后在85℃下热灭活5分钟。
    4. 通过15个循环的低周期PCR扩增(在98℃初始变性30秒; 15个循环的98℃10秒,60℃30秒,和72℃15秒),来富集cDNA;随后在72℃最终延伸10分钟)以产生用于深度测序的完整模板并使用聚丙烯酰胺凝胶清洁,随后使用糖原,3M乙酸钠和无水乙醇进行过夜凝胶洗脱和盐沉淀。将沉淀物重新悬浮在不含核酸酶的水中。
    5. 使用Qubit荧光计(50 ng /μl)定量准备的文库,并通过在高灵敏度DNA试剂盒上运行等分试样来验证质量。


      图3。 TruSeq小RNA样品制备流程图

    6. 使用软件/程序(如cutadapt-0.9.3(Martin,2011),bowtie-0.12.7(Langmead和Salzberg,2012),SeqQCv2.1(DeLuca等人,2012))分析小RNA,和BLAST(Altschul等人,1990)。分析步骤如下:
      1. 排序示例读取文件
        从GAIIx排序生成fastq文件,并用于下游分析。
      2. 序列读取质量控制(QC)报告
        对来自不同泳道的不同样品的所有产生的序列读数进行QC(Mugasimangalam等人,正在制备的手稿)。基于高质量读数的百分比来考虑所有测序读取的QC报告,读取中70%的碱基具有> 20 phred得分。基于根据QC(HQ读数> 90%)的分析,报告过程进一步测序
      3. 修剪读取
        使用cutadapt-0.9.3进行修整以从原始小RNA序列中除去连接的接头序列。 Cutadapt工具从fastq文件中删除用户指定的序列(Martin,2011)。
      4. 生成唯一的读取和计数
        通过编写自定义脚本并生成读取计数来识别唯一读取。下面简要描述的步骤进行:
        删除长度小于或等于15个碱基的读数。考虑其长度在16至36个碱基范围内的读数。识别唯一序列并删除重复序列。通过确定序列数据中存在多少次,为每个唯一读取生成读取计数。
      5. SnoRNA,SnRNA,rRNA,tRNA的注释
        使用bowtie-0.12.7将序列与大豆基因组比对。 Bowtie支持fastq读取到参考fasta文件的端对端比对(Langmead等人,2009)。端到端比对仅考虑那些与参考fasta文件完全与3个错配匹配的读数。使用BLASTn(Altschul等人,1990)针对Rfam数据库(使用默认参数)检查与多达3个错配匹配的读数,以鉴定测序数据中的其他小RNA(Meyers等人,/em>。,2008)。
      6. miRNA注释
        检查由BLASTn(E值截止为10)的唯一序列对miRbase数据库以鉴定已知的miRNA。 Blast基于参考和查询之间的序列同源性(唯一读取)来识别相似的序列

食谱

  1. T4 RNA连接酶2缓冲液
    31mM Tris-HCl pH7.5
    6.3mM MgCl 2 v/v 0.63mM DTT
    0.2 mM ATP
    5.2%PEG8000

致谢

该协议的原始版本在Paul等人中描述。 (2014年)。这项工作得到印度新德里科学和工业研究理事会荣誉名誉科学家项目[第21(0884)/12/EMR-II号]的支持。

参考文献

  1. Altschul,SF,Gish,W.,Miller,W.,Myers,EW和Lipman,DJ(1990)。  基本局部比对搜索工具 215(3):403-410。
  2. DeLuca,DS,Levin,JZ,Sivachenko,A.,Fennell,T.,Nazaire,MD,Williams,C.,Reich,M.,Winckler,W。和Getz,G。(2012) ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22539670"target ="_ blank"> RNA-SeQC:用于质量控制和过程优化的RNA-seq指标。 a> Bioinformatics 28(11):1530-1532。
  3. Langmead,B.,Trapnell,C.,Pop,M.and Salzberg,SL(2009)。  短DNA序列与人类基因组的超快速和记忆效率比对。基因组生物学 10(3):R25。
  4. Langmead,B。和Salzberg,SL(2012)。  使用Bowtie 2的快速间隙读取校准。 Nat Methods 9(4):357-359。
  5. Martin,M.(2011)。  Cutadapt删除适配器序列从高通量测序读取。 EMB J 17(1):第10-12页。
  6. Meyers,BC,Axtell,MJ,Bartel,B.,Bartel,DP,Baulcombe,D.,Bowman,JL,Cao,X.,Carrington,JC,Chen,X.,Green,PJ,Griffiths-Jones, ,Jacobsen,SE,Mallory,AC,Martienssen,RA,Poethig,RS,Qi,Y.,Vaucheret,H.,Voinnet,O.,Watanabe,Y.,Weigel,D。和Zhu,JK(2008) ; 植物MicroRNA的注释标准 Plant Cell 20(12):3186-3190。
  7. Mugasimangalam,R.,Mohan,K.,Jayakumar,V.,Ashick,M. M.,Kapila,G.and Niranjan,V.SeqQC:rapid quality control of next generation sequence data(正在准备的手稿)。
  8. Paul,S.,Kundu,A。和Pal,A。(2014)。  15-23。
  9. TruSeqsmall RNA样品制备。 Illumina公司。
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引用:Kundu, A., Paul, S., Pal, A. and Technology, G. (2016). Mungbean Yellow Mosaic India Virus (MYMIV)-infection, Small RNA Library Construction and Deep Sequencing for MicroRNA Identification in Vigna mungo. Bio-protocol 6(20): e1961. DOI: 10.21769/BioProtoc.1961.
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