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Human, Bacterial and Fungal Amplicon Collection and Processing for Sequencing
人、细菌和真菌扩增子的采集、处理并测序   

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Abstract

Sequencing taxonomic marker genes is a powerful tool to interrogate the composition of microbial communities. For example, bacterial and fungal community composition can be evaluated in parallel using the 16S ribosomal RNA gene for bacteria or the internal transcribed spacer region in fungi. These are conserved regions that are universal to a taxonomic clade, yet have undergone some degree of evolution such that different lineages can be differentiated. Conserved regions are used for design of universal priming sites that allow amplification of the marker gene out of a mixed microbial community. Here, we describe our standard operating procedure to collect and sequence 16S rRNA and ITS1 amplicons from human skin. We use the 16S rRNA V1-V3 region for skin samples, as it has greater power for classifying common staphylococci in the skin. This protocol is adapted for 454 pyrosequencing of amplicons.

Keywords: Microbiome(微生物), 16S rRNA(16S rRNA), ITS(智能交通系统), Skin microbiome extraction(皮肤微生物提取)

Materials and Reagents

  1. Sample collection and DNA extraction
    1. Catch-All sample collection swabs (Epicentre, catalog number: QEC89100 )
    2. MasterPure yeast DNA purification kit (Epicentre, catalog number: MPY80200 )
    3. Yeast cell lysis buffer (Epicentre, catalog number: MPY80200)
    4. ReadyLyse (Epicentre, catalog number: R1810M )
    5. Promega DNA IQ Spin baskets (Promega Corporation, catalog number: U1221 )
    6. Stainless steel beads, 5mm (QIAGEN, catalog number: 69989 )
    7. MoBio PCR water (MoBio, catalog number: 17000-10 )
    8. 100% (200 proof) ethanol (Warner-Graham Company, catalog number: 64-17-5 )
    9. 70% ethanol made from 350 ml of 100% ethyl alcohol and 150 ml of MoBio water
    10. PureLink Genomic DNA Mini Kit (Life Technologies, InvitrogenTM, catalog number: K182002 )

  2. PCR amplification
    1. Accuprime Taq polymerase HiFi (Life Technologies, InvitrogenTM, catalog number: 12346-086 )
    2. Forward and reverse primers with designated barcodes (IDT custom order)
    3. MinElute PCR purification kit (QIAGEN, catalog number: 28006 )
    4. QuantIT ds DNA assay, high sensitivity (Life Technologies, InvitrogenTM, catalog number: P7589 )
    5. Ampure (SPRI) beads, 60 ml kit (Agencourt, catalog number: A63881 )
    6. TE (pH 8.0) (Life Technologies, catalog number: AM9849 )

Equipment

  1. 2.0 ml Safe-Lock Biopur individually sealed tubes (Eppendorf, catalog number: 0030 121.597 )
  2. Sterile scissors (VWR, catalog number: 82027-594 )
  3. Sterile tweezers (VWR, catalog number: 231-SA-SE )
  4. Safe-Lock PCR Biopur tube (Eppendorf, catalog number: 0030 123.344 )
  5. 15 and 50 ml conical tubes (Falcon, catalog numbers: 14-959-49D and 14-432-22 )
  6. 96-well thermocycler plates (USA Scientific, catalog number: 1402-9596 )
  7. Clear adhesive plate seals (USA Scientific, catalog number: 2978-2100 )
  8. Foil plate seals (USA Scientific, catalog number: 2923-0110 )
  9. Reagent reservoirs (USA Scientific, catalog number: 2320-2620 )
  10. Heated shaking block that holds Eppendorf tubes (Eppendorf Thermomixer C)
  11. Bead beater (Qiagen TissueLyser II, catalog number: 85300 )
  12. Bead beater adapters (Qiagen TissueLyser Adapter Sets, catalog number: 69982 )
  13. UV crosslinker (UVP, catalog number: CL-1000 )
  14. Microcentrifuge (Eppendorf, Centrifuge 5415D )
  15. Thermocycler (Applied Biosystems Veriti)
  16. Plate centrifuge (Beckman Coulter, Allegra 6KR)
  17. Fluorometer plate reader (Thermo Scientific, catalog number: 5210450 )
  18. Multi-channel pipettes (pre- and post-PCR designated)
  19. 96- well microplates for fluorescence-based assays (Life Technologies, catalog number: M33089 )
  20. Qubit 2.0 Fluorometer (Life Technologies, InvitrogenTM, catalog number: Q32866 )
  21. Qubit 2.0 Quantitation Starter Kit (Life Technologies, InvitrogenTM, catalog number: Q32871 )

Procedure

  1. Sample collection
    1. Add 100 μl of yeast cell lysis buffer to a 2 ml safe-lock tube.
    2. Open the swab pack, pre-moisten swab in lysis buffer, and then swab the selected skin site with the swab for 10 sec. For high-resolution skin sampling, a 4 cm2 skin area is swabbed. Then return the swab to lysis buffer and with sterile scissors, cut off the swab so that the top is even with the swab stem so that it will fit in the tube. If the swab is cut too short, recovery during the DNA extraction step is more difficult. Collect a negative control by waving a pre-moistened swab in the air, then placing into the lysis buffer.
    3. Samples in lysis buffer can be stored at -80 °C prior to processing. While it is unknown whether samples are stable indefinitely, good results have been obtained from samples frozen for several years.

  2. DNA extraction
    1. Clean bench and centrifuge with 70% ethanol and 10% bleach. UV-treat pipettes and tube racks for 30 min prior to use.
    2. Thaw tubes for 10 min at 37 °C on heated shaking block.
    3. Short spin tubes, then open and add 1 μl of ReadyLyse and 250 μl of Yeast Cell Lysis Solution.
    4. Incubate tubes for 60 min at 37 °C on heated shaking block.
    5. During this time, UV-treat for 30 min an appropriate number of Promega IQ Spin baskets and stainless steel beads, for example by placing them in a sterile, covered petri dish. Also UV-treat a pair of tweezers.
    6. After incubation, short spin tubes.
    7. Pick up a Promega basket with tweezers one hand, open a tube with the other hand. Take the basket with the other hand and then use the tweezers to pick up the tip of the swab. While holding the swab, place the basket into the tube, then replace the swab into the tube+basket. Between samples, wipe the tweezers well with a paper towel soaked with 70% ethanol.
    8. Place the open tube + basket + swab into a centrifuge and short spin to collect excess buffer. Remove basket + swab and discard.
    9. With tweezers wiped down with 70% ethanol, open the tube and add two 5-mm stainless steel beads to each sample taking care not to touch the tweezers to the tube. Close tubes and load tubes into the QIAGEN bead beater adaptor and balance. Beat for 2 min at 30 Hz.
    10. Removed tubes and incubate for 30 min at 65 °C on heated shaking block.
    11. Place on ice for 5 min. Short spin tubes.
    12. Add 150 μl Epicentre MPC Reagent. Vortex tube 10 sec, then spin 10 min. In the meanwhile, label a fresh set of 2 ml Eppendorf tubes.
    13. Transfer the supernatant, being careful not to disturb the pellet at the bottom of the tube, to the fresh tube and add an equal volume of 100% EtOH. Mix by inverting several times.
    14. Set up PureLink Genomic DNA Mini Kit genomic DNA extraction columns with their collection tubes according to manufacturer’s instructions. Apply sample to the extraction column. Spin at maximum speed for 1 min. Remove flow-through and repeat if sample remains. Place column into a fresh collection tube.
    15. Wash column with 500 μl Buffer 1, spin at maximum speed for 1 min, discard flow-through, and place column into a fresh collection tube.
    16. Wash column with 500 μl Buffer 2. Spin at maximum speed for 1 min, discard flow-through, and then spin at maximum speed for 3 min to dry the column.
    17. Discard flow-through and place column in clean safe-lock PCR tube. Add 25 μl ultrapure PCR water and let stand for 3 min. Spin at maximum speed for 30 sec to elute. Discard column and store eluate at -20 °C.
    18. Resultant genomic DNA is typically quantified according to manufacturer’s instructions with Qubit fluorometric quantitation. Regardless of concentration, 2 μl per sample is used for subsequent amplification.

  3. 16S amplification of V1-V3 region for 454 sequencing
    1. Here, 2 μl of eluate per reaction is used, and reactions are performed in duplicate and combined prior to purification. The concentration of the eluate is highly variable depending on sample origin. For example, low abundance skin sites can yield under 1 ng/μl, while high abundance skin sites can yield greater than 50 ng/μl.
      Primer V1_27F: 5’-AGAG TTTGATCCTGGCTCAG-3’
      Primer V3_534R w/ barcodes: 5’-ATTACCGCGGCTGCTGG-3’
      Primers to other regions, and sample barcodes, are available online at http://www.hmpdacc.org.
    2. To create the premix for two 96-well plates, combine buffer, Taq, forward primer, and water into a 15 ml conical tube and vortex to mix. Keep on ice. All amounts are in μl.


      1 reaction
      110x reactions
      10x Accuprime buffer II
      2
      220
      Accuprime taq
      0.15
      16.5
      100 μM Primer V1_27F
      0.05
      2.2
      2 μM Primer V3_534R with barcode
      2
      -
      DNA eluate
      2
      -
      Water (for PCR)
      13.8
      1519.1

    3. Aliquot 23 μl of premix into each well of two 96-well plates.
    4. Add the barcoded reverse primer to each well.
    5. Add DNA to each well.
    6. Seal plates with thermofoil, and spin down.
    7. Run the following PCR program:
      95 °C, 2 min
      30 cycles of:
      95 °C, 20 sec
      56 °C, 30 sec
      72 °C, 5 min
      Then,
      4 °C, forever

  4. ITS1 amplification for 454 sequencing
    1. Here, 4 μl of eluate per reaction is used, and reactions are performed in duplicate and combined prior to purification.
      Primer ITS1F: 5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGTAAAAG TCGTAACAAGGTTTC-3’
      Primer ITS1R with barcodes: 5’- GTTCAAAGAYTCGATGATTCAC-3’
    2. To create the premix for two 96-well plates, combine buffer, Taq, forward primer, and water into a 15 ml concial tube and vortex to mix. Keep on ice. All amounts are in μl.
       

      1 reaction
      110x reactions
      10x Accuprime buffer II
      2.5
      275
      Accuprime taq
      0.2
      22
      100 μM Primer ITSF
      0.1
      11
      Water (for PCR)
      16.2
      2002



      2 μM Primer ITS1R with barcode
      2
      -
      DNA eluate
      4
      -

    3. Aliquot 19 μl of premix into each well of two 96-well plates.
    4. Add the barcoded reverse primer to each well.
    5. Add DNA to each well.
    6. Seal plates with foil plate seals, and spin down.
    7. Run the following PCR program:
      95 °C, 2 min
      32 cycles of:
      95 °C, 30 sec
      50 °C, 30 sec
      72 °C, 2 min
      Then,
      72 °C, 5 min
      4 °C, forever

  5. PCR Clean-up with Agencourt SPRI kit
    1. Spin down plates, then combine V1-V3 reactions into one plate, and ITS reactions into one plate.
    2. Add 72 μl Agencourt Beads to each well, pipetting up and down 10x.
    3. Incubate at room temperature for 5 min to bind DNA to beads.
    4. Place plate on magnet for 2 min to separate beads from solution.
    5. Keeping the plate on the magnet, aspirate cleared solution from reaction plate (take care to avoid aspirating beads on the bottom of the wells), and discard.
    6. Keeping the plate on the magnet, add 200 μl 70% ethanol to each well. Aspirate. Repeat.
    7. Keep the plate open for 5 min to dry beads.
    8. Remove the plate off the magnet and add 25 μl TE, pH 8.0 to the plate. Allow to elute for 1 min.
    9. Place plate back on magnet and allow beads to separate for 1 min.
    10. Transfer eluate to a new plate.

  6. PCR product quantification using QuantIT dsDNA high-sensitivity assay kit
    1. Bring kit components to room temperature.
    2. For two plates, mix 40 ml of QuantIT buffer with 200 μl of QuantIT reagent in a 50 ml conical tube.
    3. Aliquot 200 μl of buffer-reagent mix to each well of two 96 well microplates for fluorescence-based assays.
    4. Add 10 μl of each standard from the kit (8 total standards at 0, 0.5, 1, 2, 4, 6, 8, and 10 ng/μl) to first two columns on each plate. Standards are read in duplicate and samples a single time.
    5. Add 2 μl of PCR products to wells, leaving a column of blanks between the standards and samples.
    6. Including a 10 sec shake step, measure fluorescence on Thermo plate reader with filter pair 485/538.
    7. Generate standard curve from standards, subtracting background.
    8. Calculate PCR product concentration from standard curve and equation.

  7. PCR product pooling and purification
    1. Pool approximately equal amounts of each PCR product into a single Eppendorf tube. The target ng per sample is determined by the lowest concentration sample (excluding controls). For the lowest concentration sample(s), add the entire PCR product, and for those with higher amounts, add an approximately equivalent amount.
    2. Purify pool with QIAGEN minElute column according to manufacturer’s instructions. Briefly, add 5 volumes of buffer PB to 1 volume of the combined PCR pool.
    3. Apply the sample to the MinElute column that has been placed in a 2 ml collection tube.
    4. To bind DNA, centrifuge 30 sec at maximum speed. Discard flowthrough and place the column back in the same tube.
    5. Wash with 750 μl buffer PE, discard flowthrough, then centrifuge for an additional 1 min.
    6. Place the column into a fresh tube and elute in 30 μl TE (pH 8.0). This final product represents an approximately equivalent concentration of each of the 96 input samples. To ensure quality, one may then quantitate the pooled library according to manufacturer’s instructions with Qubit fluorometric quantitation, or by running 2 μl on an electrophoretic agarose gel or equivalent. For the V1-V3 amplicon, the product size should fall between 450-550 bp.
    7. Single, pooled amplicon libraries in an Eppendorf tubes can then be sent to a sequencing facility for sequencing on a 454 GS FLX (Roche, http://454.com/products/gs-flx-system/index.asp) instrument using titanium chemistry.

Representative data

  1. For sequence processing, we recommend using the mothur software (http://www.mothur.org) together with the 454 standard operating protocol written by Pat Schloss (http://www.mothur.org/wiki/454_SOP):
  2. In summary, the first purpose of sequence processing is deconvolutes the raw 454 data into individual samples and then to remove poor quality sequence arising from technical artifacts produced by PCR or 454 sequencing error. The second step of sequence processing is then to make sense of the amplicon sequences. For studies based on 16S rRNA amplicon sequencing, sequences are processed into operational taxonomic units (OTUs) that represent a bacterial or archaeal ‘species’. For fungal studies based on the ITS, we prefer to classify sequences into ‘phylotypes’, as sequence distance that is the basis of the formation of OTUs is very variable for the ITS, which is under very different selective pressure than the 16S rRNA, and depends on the fungal taxon. For species level classification of genera of interest, we created custom taxonomic references using the pplacer algorithm, written by Eric Matsen (http://matsen.fhcrc.org/pplacer). For example, to speciate staphylococcal sequences, 16S full-length rRNA sequences belonging to different staphylococcal species can be downloaded from Genbank or RDP (http://rdp.cme.msu.edu) for construction of the phylogenetic tree. It is important to use high quality (e.g., type strains, fully sequenced genomes) to construct the phylogenetic model.
  3. We then use the R software to post-process files generated by mothur. For example, to generate plots of the relative abundances between species, we use the *.tax.summary file generated by the classify.seqs() or the classify.otu() command in mothur (Figure 1). A sample R script with sample data are provided at https://github.com/julia0h/amplicon_bioprotocol.git in the plottingTaxonomy/ folder.
  4. For examining alpha diversity in the sample, we typically use the Shannon diversity index, Inverse Simpson, or species observed from the summary.single() command in mothur (Figure 2). Sample scripts are provided in plottingDiversity/.
  5. Finally, for comparing samples with a distance metric like the Yue-Clayton theta index to generate principal coordinate analysis plots, we use the output of the dist.shared() and pcoa() commands (Figure 3). Sample scripts are provided in plottingPCOA/.


    Figure 1. Example relative abundance plots of a control vs disease group


    Figure 2. Example alpha diversity analysis looking at the differences in Shannon diversity at 3 different sites that are/are not treated


    Figure 3. Example principle coordinates analysis based on Yue-Clayton theta values that show the similarity between samples from different individuals. Taxa contributing significantly to variation between samples are shown with lines.

Acknowledgments

This protocol is based on the data generated and analyses performed in Oh et al. (2013). Skin sampling procedures were designed by Dr. Heidi Kong, and DNA extraction and amplification procedures by Clayton Deming, Cynthia Ng, and Elizabeth Grice at the National Institutes of Health.

References

  1. Oh, J., Freeman, A. F., Program, N. C. S., Park, M., Sokolic, R., Candotti, F., Holland, S. M., Segre, J. A. and Kong, H. H. (2013). The altered landscape of the human skin microbiome in patients with primary immunodeficiencies. Genome Res 23(12): 2103-2114.
  2. Schloss, P. D., Westcott, S. L., Ryabin, T., Hall, J. R., Hartmann, M., Hollister, E. B., Lesniewski, R. A., Oakley, B. B., Parks, D. H., Robinson, C. J., Sahl, J. W., Stres, B., Thallinger, G. G., Van Horn, D. J. and Weber, C. F. (2009). Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75(23): 7537-7541.
  3. Schloss, P. D., Gevers, D. and Westcott, S. L. (2011). Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One 6(12): e27310.
  4. Matsen, F. A., Kodner, R. B. and Armbrust, E. V. (2010). pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC Bioinformatics 11: 538.

简介

测序分类标记基因是一个强有力的工具,以询问微生物群落的组成。 例如,可以使用细菌的16S核糖体RNA基因或真菌中的内部转录间隔区平行评估细菌和真菌群落组成。 这些是对分类学分支是通用的保守区,但已经经历了某种程度的进化,使得不同谱系可以分化。 保守区用于设计允许从混合微生物群落扩增标记基因的通用引发位点。 在这里,我们描述我们的标准操作程序收集和序列16S rRNA和ITS1扩增子从人类皮肤。 我们使用16S rRNA V1-V3区域的皮肤样本,因为它有更大的权力分类皮肤中的常见葡萄球菌。 该方案适用于扩增子的454焦磷酸测序。

关键字:微生物, 16S rRNA, 智能交通系统, 皮肤微生物提取

材料和试剂

  1. 样品收集和DNA提取
    1. Catch-所有样品收集拭子(Epicentre,目录号:QEC89100)
    2. MasterPure酵母DNA纯化试剂盒(Epicentre,目录号:MPY80200)
    3. 酵母细胞裂解缓冲液(Epicentre,目录号:MPY80200)
    4. ReadyLyse(Epicentre,目录号:R1810M)
    5. Promega DNA IQ旋转篮(Promega Corporation,目录号:U1221)
    6. 不锈钢珠,5mm(QIAGEN,目录号:69989)
    7. MoBio PCR水(MoBio,目录号:17000-10)
    8. 100%(200标准)乙醇(Warner-Graham公司,目录号:64-17-5)
    9. 由350ml 100%乙醇和150ml MoBio水制成的70%乙醇
    10. PureLink Genomic DNA Mini Kit(Life Technologies,Invitrogen TM,目录号:K182002)

  2. PCR扩增
    1. Accuprime Taq聚合酶HiFi(Life Technologies,Invitrogen TM ,目录号:12346-086)
    2. 带指定条形码的正向和反向引物(IDT自定义顺序)
    3. MinElute PCR纯化试剂盒(QIAGEN,目录号:28006)
    4. QuantIT ds DNA测定,高灵敏度(Life Technologies,Invitrogen TM ,目录号:P7589)
    5. Ampure(SPRI)珠,60ml试剂盒(Agencourt,目录号:A63881)
    6. TE(pH8.0)(Life Technologies,目录号:AM9849)

设备

  1. 2.0ml Safe-Lock Biopur单独密封管(Eppendorf,目录号:0030121.597)
  2. 无菌剪刀(VWR,目录号:82027-594)
  3. 无菌镊子(VWR,目录号:231-SA-SE)
  4. 安全锁定PCR Biopur管(Eppendorf,目录号:0030123.344)
  5. 15和50ml锥形管(Falcon,目录号:14-959-49D和14-432-22)
  6. 96孔热循环板(USA Scientific,目录号:1402-9596)
  7. 透明粘性板密封件(美国科学,目录号:2978-2100)
  8. 箔板密封(美国科学,目录号:2923-0110)
  9. 试剂库(USA Scientific,目录号:2320-2620)
  10. 加热震动块,装有Eppendorf管(Eppendorf Thermomixer C)
  11. Bead beater(Qiagen TissueLyser II,目录号:85300)
  12. 珠子搅拌器适配器(Qiagen TissueLyser Adapter Sets,目录号:69982)
  13. UV交联剂(UVP,目录号:CL-1000)
  14. 微量离心机(Eppendorf,Centrifuge 5415D)
  15. 热循环仪(Applied Biosystems Veriti)
  16. 板式离心机(Beckman Coulter,Allegra 6KR)
  17. 荧光计读板仪(Thermo Scientific,目录号:5210450)
  18. 多通道移液器(指定PCR前和PCR后)
  19. 用于基于荧光的测定的96孔微量培养板(Life Technologies,目录号:M33089)
  20. Qubit 2.0荧光计(Life Technologies,Invitrogen TM ,目录号:Q32866)
  21. Qubit 2.0定量起始试剂盒(Life Technologies,Invitrogen TM ,目录号:Q32871)

程序

  1. 样品收集
    1. 加入100微升酵母细胞裂解缓冲液到2毫升安全锁管。
    2. 打开拭子包,预先润湿拭子在裂解缓冲液,然后拭拭   用拭子选择皮肤部位10秒。 用于高分辨率皮肤 取样,擦拭4cm 2皮肤区域。 然后将棉签返回至裂解 缓冲液和用无菌剪刀,切掉拭子,使顶部 甚至与拭子茎,使其将适合在管中。 如果是棉签 切割太短,DNA提取步骤中的回收更多 难。 通过挥动预湿润的拭子收集阴性对照 空气,然后放入裂解缓冲液
    3. 裂解中的样品 缓冲液可以在加工前储存在-80℃。 虽然它是未知的 无论样品是否稳定,已经获得了良好的结果   从冷冻几年的样品。

  2. DNA提取
    1. 清洁工作台并用70%乙醇和10%漂白剂离心。 紫外线处理移液器和管架使用前30分钟。
    2. 在37℃下在加热的振荡块上解冻管子10分钟
    3. 短离心管,然后打开,加入1μlReadyLyse和250μl酵母细胞裂解液
    4. 孵育管在37℃下60分钟在加热的摇动块
    5. 在此期间,UV处理30分钟适当数量 Promega IQ旋转篮和不锈钢珠,例如 将它们放置在无菌的覆盖的培养皿中。 也UV处理一对 镊子。
    6. 孵育后,短旋转管。
    7. 拿起a Promega篮子用镊子一只手,用另一只手打开一根管子。 用另一只手拿着篮子,然后用镊子拿起   棉签的尖端。 拿着棉签,把篮子放进去 管,然后将拭子更换为管+篮。 在样品之间,擦拭 镊子用70%乙醇浸泡的纸巾
    8. 将开放管+篮+棉签放入离心机,短暂旋转 收集多余的缓冲液。 取出篮+棉签并丢弃。
    9. 与 镊子用70%乙醇擦拭,打开管子,加入两个5毫米 不锈钢珠对每个样品小心不要触摸 镊子到管。 关闭管和加载管到QIAGEN珠 搅拌器适配器和平衡。 在30 Hz下打2分钟。
    10. 移除管并在65℃在加热的振荡块上孵育30分钟。
    11. 置于冰上5分钟。 短旋管。
    12. 加入150μlEpicenter MPC试剂。 涡旋管10秒,然后旋转10 min。 同时,标记一套新鲜的2毫升Eppendorf管。
    13. 转移上清液,小心不要打扰沉淀   管的底部,到新鲜管并加入等体积 100%EtOH。 混合反转几次。
    14. 设置PureLink 基因组DNA Mini Kit基因组DNA提取柱及其 收集管根据制造商的说明。 应用样品 到萃取柱。 以最大速度旋转1分钟。 去掉 流过,如果样品残留则重复。 将柱子放入新鲜 收集管。
    15. 用500μlBuffer 1洗柱,旋转 最大速度1分钟,丢弃流过,并将柱放入a 新鲜采集管。
    16. 用500μl缓冲液洗涤柱2.旋转   最大速度1分钟,丢弃流过,然后最大旋转   速度3分钟以干燥色谱柱。
    17. 丢弃流通和 放置在干净的安全锁定PCR管中。 加入25μl超纯水 并静置3分钟。 旋转在最大速度30秒洗脱。 弃去色谱柱,在-20°C下保存洗脱液。
    18. 结果基因组 通常根据制造商的说明书来定量DNA 与Qubit荧光定量。 无论浓度,2微升 每个样品用于随后的扩增。

  3. 用于454测序的V1-V3区域的16S扩增
    1. 这里,使用每个反应2μl洗脱液,并进行反应 一式两份并在纯化前合并。 浓度 洗脱液是高度可变的,取决于样品来源。 例如, 低丰度的皮肤部位可以产生低于1ng /μl,而高丰度 皮肤部位可产生大于50ng /μl 引物V1_27F:5'-AGAG TTTGATCCTGGCTCAG-3'
      引物V3_534R w /条形码:5'-ATTACCGCGGCTGCTGG-3'
      可以在 http://www.hmpdacc.org上在线获取其他地区的入门指南和示例条码。
    2. 为了产生两个96孔板的预混合物,组合缓冲液,Taq, 正向引物和水倒入15ml锥形管中并涡旋混合。 保持在冰上。 所有量以μl计。


      1反应
      110x反应
      10x Accuprime缓冲区II
      2
      220
      Accuprime taq
      0.15
      16.5
      100μMPrimer V1_27F
      0.05
      2.2
      带有条形码的2μMPrimer V3_534R
      2
      -
      DNA洗脱液
      2
      -
      水(用于PCR)
      13.8
      1519.1

    3. 等分23微升预混到两个96孔板的每个孔。
    4. 向每个孔中加入条形码反向引物。
    5. 向每个孔中加入DNA。
    6. 密封板用thermofoil,并向下旋转。
    7. 运行以下PCR程序:
      95℃,2分钟
      30个循环:
      95°C,20秒
      56℃,30秒
      72℃,5分钟
      然后,
      4°C,永远

  4. 用于454测序的ITS1扩增
    1. 这里,使用每个反应4μl洗脱液,并且一式两份进行反应,并在纯化前合并。
      引物ITS1F:5'-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGTAAAAG TCGTAACAAGGTTTC-3'
      带有条形码的引物ITS1R:5'-GTTCAAAGAYTCGATGATTCAC-3'
    2. 为了产生两个96孔板的预混合物,组合缓冲液,Taq, 正向引物和水倒入15ml锥形管中并涡旋混合。 保持在冰上。 所有量以μl计。
       

      1反应
      110x反应
      10x Accuprime缓冲区II
      2.5
      275
      Accuprime taq
      0.2
      22
      100μM引物ITSF
      0.1
      11
      水(用于PCR)
      16.2
      2002



      2μM带条形码的引物ITS1R
      2
      -
      DNA洗脱液
      4
      -

    3. 等分19μl预混合到两个96孔板的每个孔。
    4. 向每个孔中加入条形码反向引物。
    5. 向每个孔中加入DNA。
    6. 密封板用铝箔密封,并向下旋转。
    7. 运行以下PCR程序:
      95℃,2分钟
      32个循环:
      95℃,30秒
      50℃,30秒
      72℃,2分钟
      然后,
      72℃,5分钟
      4°C,永远

  5. 用Agencourt SPRI试剂盒进行PCR清洗
    1. 旋转板,然后将V1-V3反应合并成一个板,将ITS反应合并成一个板
    2. 每孔加入72μlAgencourt珠,上下移动10x
    3. 在室温下孵育5分钟以将DNA结合到珠子上
    4. 将板放在磁铁上2分钟,以从溶液中分离珠子
    5. 保持板在磁铁上,吸出清除溶液 反应板(注意避免吸液珠底部   井),并丢弃
    6. 保持板在磁铁上,添加200微升70%乙醇的每个孔。 吸出。 重复。
    7. 保持板开放5分钟干珠。
    8. 从磁铁上取下板,加入25μlTE,pH 8.0的板。 允许洗脱1分钟。
    9. 将板放回磁铁,让珠子分开1分钟。
    10. 将洗脱液转移至新平板。

  6. 使用QuantIT dsDNA高灵敏度测定试剂盒进行PCR产物定量
    1. 将套件部件的温度升至室温。
    2. 对于两个板,在50ml锥形管中混合40ml的QuantIT缓冲液与200μl的QuantIT试剂。
    3. 等分200微升缓冲试剂混合物到两个96孔微量培养板的每个孔用于基于荧光的测定
    4. 从试剂盒中添加10μl的每个标准品(8总标准在0, 0.5,1,2,4,6,8和10 ng /μl)。 标准重复读取,并单次采样。
    5. 向孔中加入2μlPCR产物,在标准品和样品之间留下一列空白。
    6. 包括10秒振荡步骤,在具有过滤器对485/538的Thermo板读数器上测量荧光。
    7. 从标准生成标准曲线,减去背景。
    8. 从标准曲线和方程计算PCR产物浓度。

  7. PCR产物合并和纯化
    1. 将大约等量的每种PCR产物合并成单个 Eppendorf管。 每个样品的目标ng由最低确定 浓度样品(不含对照)。 对于最低浓度 样品,添加整个PCR产物,以及对于具有更高的PCR产物 金额,添加大致相等的金额。
    2. 净化池 用QIAGEN minElute柱根据制造商的说明书。 简言之,向1体积的组合PCR中加入5体积的缓冲液PB 池。
    3. 将样品应用于已置于2 ml收集管中的MinElute色谱柱。
    4. 为了结合DNA,以最大速度离心30秒。 弃去流出物并将柱放回同一管中。
    5. 用750μl缓冲液PE洗涤,弃去流出液,然后离心1分钟
    6. 将柱置于新管中,并在30μlTE(pH 8.0)中洗脱。 该最终产物表示近似相等的浓度 的96个输入样本中的每一个。 为了确保质量,可以然后 根据制造商的说明定量合并的文库 用Qubit荧光定量,或通过在2μl上运行 电泳琼脂糖凝胶或等同物。 对于V1-V3扩增子, 产品尺寸应在450-550 bp之间。
    7. 单人,合租 然后可以将Eppendorf管中的扩增子文库发送至 用于在454GS FLX上测序的测序装置(Roche, http://454.com /products/gs-flx-system/index.asp )仪器使用 钛化学。

代表数据

  1. 对于序列处理,我们建议您使用mothur软件( http://www.mothur.org )和454由Pat Schloss(http://www.mothur.org/wiki/454_SOP)编写的标准操作协议:
  2. 总之,序列处理的第一目的是将原始454数据去卷积为单个样本,然后去除由于PCR或454测序错误产生的技术伪像导致的质量差的序列。然后序列处理的第二步是使扩增子序列有意义。对于基于16S rRNA扩增子测序的研究,将序列加工成代表细菌或古细菌"物种"的操作分类单位(OTU)。对于基于ITS的真菌研究,我们优选将序列分类为"系统型",因为作为形成OTU的基础的序列距离对于ITS是非常可变的,其处于与16S rRNA非常不同的选择压力下,取决于真菌分类群。对于感兴趣的物种水平分类,我们使用由Eric Matsen编写的pplacer算法创建了自定义分类参考( http://matsen.fhcrc.org/pplacer )。例如,为了鉴定葡萄球菌序列,属于不同葡萄球菌种的16S全长rRNA序列可以从Genbank或RDP( http://rdp.cme.msu.edu )构建系统发育树。使用高质量(例如,型菌株,完全测序的基因组)构建系统发生模型是重要的。
  3. 然后我们使用R软件对mothur生成的文件进行后处理。例如,为了生成物种之间的相对丰度的图,我们使用在mothur(图1)中由classify.seqs()或classify.otu()命令生成的* .tax.summary文件。在 https://github.com/julia0h/amplicon_bioprotocol.git在plottingTaxonomy /文件夹中
  4. 为了检查样本中的α多样性,我们通常使用Shannon多样性指数,Inverse辛普森或从motuur中的summary.single()命令观察到的物种(图2)。示例脚本在plottingDiversity /中提供。
  5. 最后,为了将样本与诸如Yue-Claytonθ索引的距离度量进行比较以生成主坐标分析图,我们使用dist.shared()和pcoa()命令的输出(图3)。示例脚本在plottingPCOA /。
    中提供

    图1.对照组与疾病组的示例相对丰度图


    图2.示例alpha多样性分析,看看在3个不同网站的Shannon多样性之间的差异


    图3.基于Yue-Claytonθ值的示例原理坐标分析,显示来自不同个体的样本之间的相似性。用线显示对样本之间变化有显着影响的分类。

致谢

该协议基于在Oh等人(2013)中生成和分析的数据。皮肤取样程序由Heidi Kong博士设计,以及由Clayton Deming,Cynthia Ng和Elizabeth Grice在National Institutes of Health设计的DNA提取和扩增程序。

参考文献

  1. Oh,J.,Freeman,A.F.,Program,N.C.S.,Park,M.,Sokolic,R.,Candotti,F.,Holland,S.M.,Segre,J.A.and Kong,H.H。(2013)。 原发性免疫缺陷患者人类皮肤微生物组的改变景观 Genome Res 23(12):2103-2114。
  2. Schloss,PD,Westcott,SL,Ryabin,T.,Hall,JR,Hartmann,M.,Hollister,EB,Lesniewski,RA,Oakley,BB,Parks,DH,Robinson,CJ,Sahl,JW,Stres, ,Thallinger,GG,Van Horn,DJ和Weber,CF(2009)。 介绍mothur:开源,平台独立,社区支持的软件,用于描述和比较微生物群落。 Appl Environ Microbiol 75(23):7537-7541。
  3. Schloss,P.D.,Gevers,D.and Westcott,S.L。(2011)。 降低PCR扩增和测序工件对基于16S rRNA的研究的影响。 em> PLoS One 6(12):e27310。
  4. Matsen,F.A.,Kodner,R.B.and Armbrust,E.V。(2010)。 pplacer:序列在固定参考树上的线性时间最大似然和贝叶斯系统发生位置。 a> BMC Bioinformatics 11:538.
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Oh, J. (2015). Human, Bacterial and Fungal Amplicon Collection and Processing for Sequencing. Bio-protocol 5(10): e1477. DOI: 10.21769/BioProtoc.1477.
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Ella Chang
UCSF
Thanks Julia for putting together such a thorough protocol. I would like to use this protocol to study skin microbiome in psoriasis patients. As a practice run, I tried to isolate microbiome from my scalp, and use a sterile unused swab as a negative control. I eluted DNA with 25ul water as the protocol suggested and checked the yield using NanoDrop. Both the scalp sample and the negative control yield 17ng/ul. I am just wondering if it is a bad idea to use nanoDrop to access the quality of the prep since the amount of skin microbiome is low. If not using NanoDrop, how do you make sure the DNA extraction went well? I am also wondering if you could kindly share your experience on how much DNA you normally get using this protocol to isolate skin microbiome.

thanks a lot for your help

Ella
1/11/2016 2:11:48 PM Reply
Julia Oh
National Human Genome Research Institute, National Institutes of Health, MD

Hi Ella,

Try the high-sensitivity (not broad range) Qubit kit. We find that NanoDrop is not accurate at low concentrations.

Yield varies very significantly by skin site. For example, for the inner nostril, you might expect 50 ng/uL; for a low biomass sites like the forearm or inner elbow you might achieve 0.5 ng/uL. Run your negative control in your PCR (also include a negative control for the PCR only), and see if you get a detectable band with yield--that will be informative as to potential contamination in your collection method.

1/11/2016 2:15:58 PM


Ella Chang
UCSF

Hi Julia,

Thanks a lot for your prompt reply. This is very helpful. I will try PCR next.

1/12/2016 9:52:59 AM


Ella Chang
UCSF

Dear Julia,

I tried to run a PCR using 2ul of the microbiome elute (isolated from scalp swab and air control) and V1_27F and V3_534R primers. I also included a water control in this PCR reaction. I only saw a very faint band around 8kb in the scalp sample and both negative controls. Given that the band size is large( I was expecting a smaller size) and the faint signal of the band, I think this band might be just a background band. I am wondering if you normally see a distinct band when you do the 16S rRNA PCR. The PCR was done using the program provided in 16S amplification section in your protocol.

I think the problem for my experiment is that I didn't break up the cells enough to release DNA. According to the protocol, it looks like we are only using the PureLink genomic DNA mini kit for DNA purification (binding DNA to the column and washes with two buffers). The actual cell lysis was done using the MasterPure kit. Since this kit is designed for yeast DNA extraction, do you think it can affect the efficiency on extracting bacterial DNA? I think I will try to lyse the cells longer in the bead beater and see if I can improve the lysis. I will also really appreciate it if you have any suggestions and tips on isolating skin microbiome that I can try in my next experiment.

thanks a lot for you help.

1/15/2016 5:01:18 PM


Julia Oh
National Human Genome Research Institute, National Institutes of Health, MD

Hi Ella,

We've had good experience co-extracting bacterial DNA using this protocol (we use the same eluate for both 16S and ITS sequencing). Depending on the skin site, we may or may not see a band on the gel. High yield sites (e.g., inner nares) can have a band. Did you do a positive control? You might run a positive extraction control, like the nares. In our experience, the combination of the lysis buffer + bead beating is sufficient to lyse both bacterial and fungal cells.

1/18/2016 4:21:10 PM


Ella Chang
UCSF

Hi Julia,

Thanks a lot for your response. My samples were collected from scalp, so I guess this may not be one of the high yield sites. Thanks for the suggestion of using the inner nares as a positive control. We thought about running a positive but didn't know better about what would be a good control. Thanks again for helping me out. I really appreciate it.

1/18/2016 7:48:34 PM


Wojciech Francuzik
Charité Universitätsmedizin Berlin
1. To which reagent does the first step of the protocol "Add 100 μl of yeast cell lysis buffer to a 2 ml safe-lock tube. " refer to?

Yeast cell lysis buffer is listed in the materials and reagents list as "(Epicentre, catalog number: MPY80200)". This is actually the MasterPure Kit.

Is it a reagent from the kit?

This kit contains:
Yeast Cell Lysis Solution 60 ml
MPC Protein Precipitation Reagent 50 ml
RNase A @ 5 μg/μl 200 μl
TE Buffer 7 ml

6/3/2015 7:14:11 AM Reply
Julia Oh
National Human Genome Research Institute, National Institutes of Health, MD

Yes, this is the yeast cell lysis solution from the MasterPure Kit.

6/3/2015 2:47:02 PM


Wojciech Francuzik
Charité Universitätsmedizin Berlin

Thank you for the prompt reply.

Just to be sure: after step B3:
"Short spin tubes, then open and add 1 μl of ReadyLyse and 250 μl of Yeast Cell Lysis Solution."

we will have 350 μl of Yeast Cell Lysis Solution in our tube.

Why are we only starting with 100 μl for the extraction? 100 μl is rather small volume to moisten the Catch All swab. I would normally use 1000 μl of the SCF-1 solution (described in the methodology of the HMP for sampling skin).

Could you shortly explain the rationale here?

6/3/2015 4:04:54 PM


Julia Oh
National Human Genome Research Institute, National Institutes of Health, MD

Because of the low biomass in the skin, we try to keep the volumes low. This amount is adequate to moisten the swab for sampling a 4cm2 area. If you are planning on sampling a larger area, it'd be worth experimenting with larger volumes.

6/4/2015 10:00:56 AM