Name: a method for targeted 16s sequencing of human milk samples
Uploaded: 2018-03-23T12:30:00.000+00:00
Duration: 9 min 9 s
Description: Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

プロトコルの開発の Helty Adisetiyo 博士と Shangxin 博士はヤンに感謝したいと思います。全体的なサポートのため、国際母性小児思春期エイズ臨床試験グループ (IMPAACT) は、国立研究所のアレルギーおよび感染症 (NIAID) の国立衛生の健康 (NIH) [受賞番号によって提供されました。UM1AI068632 (IMPAACT LOC)、UM1AI068616 (IMPAACT SDMC) と UM1AI106716 (IMPAACT LC)、児童保健および人間の開発 (NICHD) のユーニス · ケネディ · シュライバーの国立研究所、国立精神衛生研究所 (NIMH) からの共同資金とします。内容は著者の責任と NIH の公式見解を必ずしも表さない。

<ol>
	<li>Ahern, P. P., Faith, J. J., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+the+human+gut+microbiota+for+effector+strains+that+shape+the+immune+system.">Mining the human gut microbiota for effector strains that shape the immune system.</a> Immunity. 40 (6), 815-823 (2014).</li><li>Postler, T. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Holobiont:+How+Microbial+Metabolites+Affect+Human+Health+and+Shape+the+Immune+System.">Understanding the Holobiont: How Microbial Metabolites Affect Human Health and Shape the Immune System.</a> Cell Metab. , (2017).</li><li>Hall, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-personal+diversity+and+temporal+dynamics+of+dental,+tongue,+and+salivary+microbiota+in+the+healthy+oral+cavity.">Inter-personal diversity and temporal dynamics of dental, tongue, and salivary microbiota in the healthy oral cavity.</a> NPJ Biofilms Microbiomes. 3, 2 (2017).</li><li>Perez Perez, G. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+Site+Is+a+More+Determinant+Factor+than+Human+Population+Diversity+in+the+Healthy+Skin+Microbiome.">Body Site Is a More Determinant Factor than Human Population Diversity in the Healthy Skin Microbiome.</a> PLoS One. 11 (4), e0151990 (2016).</li><li>Hamady, M., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+community+profiling+for+human+microbiome+projects:+Tools,+techniques,+and+challenges.">Microbial community profiling for human microbiome projects: Tools, techniques, and challenges.</a> Genome Res. 19 (7), 1141-1152 (2009).</li><li>Human Microbiome Project, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+framework+for+human+microbiome+research.">A framework for human microbiome research.</a> Nature. 486 (7402), 215-221 (2012).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+methods+and+dodging+pitfalls+in+microbiome+research.">Optimizing methods and dodging pitfalls in microbiome research.</a> Microbiome. 5 (1), 52 (2017).</li><li>Hugerth, L. W., Andersson, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+Microbial+Community+Composition+through+Amplicon+Sequencing:+From+Sampling+to+Hypothesis+Testing.">Analysing Microbial Community Composition through Amplicon Sequencing: From Sampling to Hypothesis Testing.</a> Front Microbiol. 8, 1561 (2017).</li><li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high-throughput+microbial+community+analysis+on+the+Illumina+HiSeq+and+MiSeq+platforms.">Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms.</a> ISME J. 6 (8), 1621-1624 (2012).</li><li>Pannaraj, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+Between+Breast+Milk+Bacterial+Communities+and+Establishment+and+Development+of+the+Infant+Gut+Microbiome.">Association Between Breast Milk Bacterial Communities and Establishment and Development of the Infant Gut Microbiome.</a> JAMA Pediatr. , (2017).</li><li>Ho, F. C., Wong, R. L., Lawton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+colostral+and+breast+milk+cells.+A+light+and+electron+microscopic+study.">Human colostral and breast milk cells. A light and electron microscopic study.</a> Acta Paediatr Scand. 68 (3), 389-396 (1979).</li><li>Parmely, M. J., Beer, A. E., Billingham, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+on+the+T-lymphocyte+population+of+human+milk.">In vitro studies on the T-lymphocyte population of human milk.</a> J Exp Med. 144 (2), 358-370 (1976).</li><li>Sabbaj, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus-specific+CD8(+)+T+cells+in+human+breast+milk.">Human immunodeficiency virus-specific CD8(+) T cells in human breast milk.</a> J Virol. 76 (15), 7365-7373 (2002).</li><li>Jimenez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+Analysis+of+Milk+of+Healthy+and+Mastitis-Suffering+Women.">Metagenomic Analysis of Milk of Healthy and Mastitis-Suffering Women.</a> J Hum Lact. 31 (3), 406-415 (2015).</li><li>Ghosh, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+human+immunodeficiency+virus+type+1+in+breast+milk.">Quantitation of human immunodeficiency virus type 1 in breast milk.</a> J Clin Microbiol. 41 (6), 2465-2470 (2003).</li><li>Lim, N. Y., Roco, C. A., Frostegard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+DNA/RNA+Co-extraction+Workflow+Protocol+Suitable+for+Inhibitor-Rich+Environmental+Samples+That+Focuses+on+Complete+DNA+Removal+for+Transcriptomic+Analyses.">Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses.</a> Front Microbiol. 7, 1588 (2016).</li><li>Bender, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+HIV+infection+influences+the+microbiome+of+HIV-uninfected+infants.">Maternal HIV infection influences the microbiome of HIV-uninfected infants.</a> Sci Transl Med. 8 (349), 349ra100 (2016).</li><li>Cox, M. J., Cookson, W. O., Moffatt, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+the+human+microbiome+in+health+and+disease.">Sequencing the human microbiome in health and disease.</a> Hum Mol Genet. 22 (R1), R88-R94 (2013).</li><li>Lauder, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+placenta+samples+with+contamination+controls+does+not+provide+evidence+for+a+distinct+placenta+microbiota.">Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.</a> Microbiome. 4 (1), 29 (2016).</li><li>Salter, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reagent+and+laboratory+contamination+can+critically+impact+sequence-based+microbiome+analyses.">Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.</a> BMC Biol. 12, 87 (2014).</li><li>Walker, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=16S+rRNA+gene-based+profiling+of+the+human+infant+gut+microbiota+is+strongly+influenced+by+sample+processing+and+PCR+primer+choice.">16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.</a> Microbiome. 3, 26 (2015).</li><li>Glassing, A., Dowd, S. E., Galandiuk, S., Davis, B., Chiodini, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherent+bacterial+DNA+contamination+of+extraction+and+sequencing+reagents+may+affect+interpretation+of+microbiota+in+low+bacterial+biomass+samples.">Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples.</a> Gut Pathog. 8, 24 (2016).</li><li>Kennedy, K., Hall, M. W., Lynch, M. D., Moreno-Hagelsieb, G., Neufeld, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+bias+of+illumina-based+bacterial+16S+rRNA+gene+profiles.">Evaluating bias of illumina-based bacterial 16S rRNA gene profiles.</a> Appl Environ Microbiol. 80 (18), 5717-5722 (2014).</li><li>Weiss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+down+the+sources+of+experimental+contamination+in+microbiome+studies.">Tracking down the sources of experimental contamination in microbiome studies.</a> Genome Biol. 15 (12), 564 (2014).</li><li>Aho, V. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiome+of+the+human+lower+airways:+a+next+generation+sequencing+perspective.">The microbiome of the human lower airways: a next generation sequencing perspective.</a> World Allergy Organ J. 8 (1), 23 (2015).</li><li>Charlson, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+continuity+of+bacterial+populations+in+the+healthy+human+respiratory+tract.">Topographical continuity of bacterial populations in the healthy human respiratory tract.</a> Am J Respir Crit Care Med. 184 (8), 957-963 (2011).</li><li>Bittinger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+characterization+of+medically+relevant+fungi+in+the+human+respiratory+tract+using+next-generation+sequencing.">Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing.</a> Genome Biol. 15 (10), 487 (2014).</li><li>Jervis-Bardy, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+accurate+microbiota+profiles+from+human+samples+with+low+bacterial+content+through+post-sequencing+processing+of+Illumina+MiSeq+data.">Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data.</a> Microbiome. 3, 19 (2015).</li><li>Knights, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+community-wide+culture-independent+microbial+source+tracking.">Bayesian community-wide culture-independent microbial source tracking.</a> Nat Methods. 8 (9), 761-763 (2011).</li><li>Lazarevic, V., Gaia, N., Girard, M., Schrenzel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decontamination+of+16S+rRNA+gene+amplicon+sequence+datasets+based+on+bacterial+load+assessment+by+qPCR.">Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR.</a> BMC Microbiol. 16, 73 (2016).</li><li>Kurilshikov, A., Wijmenga, C., Fu, J., Zhernakova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+Genetics+and+Gut+Microbiome:+Challenges+and+Perspectives.">Host Genetics and Gut Microbiome: Challenges and Perspectives.</a> Trends Immunol. 38 (9), 633-647 (2017).</li><li>. Mock microbial communities Available from: <a target="_blank" href="https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1">https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1</a> (2017)</li></ol>

著者が明らかに何もありません。

16S rRNA の対象となる次世代シーケンスは、マイクロバイ評価18の広く使用されている、急速な手法です。バッチの効果、環境汚染、サンプルのクロスコンタミネーション、感度、再現性など、多くの要因が悪影響実験結果に影響を与えるし、その解釈7,19を混同,20. 良い実験的なデザイン、適切なコントロ?...

人間を植民地化する微生物は人間の健康、代謝、免疫の開発、疾患感受性と予防接種や薬物療法1,への応答に影響を及ぼす疾患はとても重要と考えられて2. 現在細菌叢の人間の健康に及ぼす影響を理解するための努力を強調内のローカライズされたサイトだけでなく、定義された解剖学的なコンパートメント(すなわち皮膚、腸、口腔など)に関連付けられている微生物の同定これらのコンパートメント3,では、4が。これらの調査の努力を支え、急速な出現と増加アクセシビリティ サンプルの微生物の遺伝の内容 (マイクロバイ) の解析の並列プラットフォームを提供する次世代シーケンサー (NGS) 技術です。多くの生理学的なサンプルに関連付けられたマイクロバイは複雑で豊富な (すなわちスツール), しかし、いくつかのサンプル、マイクロバイで表される (すなわち母乳、下気道) の低いバイオマス、感度、実験的工芸品および可能な汚染の主要な問題になります。マイクロバイ研究と適切な実験的なデザインの共通の課題複数レビュー記事5,6,7,8の対象とされています。
ここに記載した NGS の堅牢な実験的パイプラインは母乳のマイクロバイを特徴付ける rRNA の 16S V4 地域9のターゲット シーケンスに基づきます。人間のミルクのマイクロバイ分析は複雑なヒトの DNA レベル背景11,12,13,14の本質的に低いバイオマス10でだけ、また高と抽出した核酸の PCR 阻害剤15,16の潜在的な持ち越し。このプロトコルは、市販の抽出キットおよびサンプル調製バッチの間で変動を最小限に抑え、半自動化されたプラットフォームに依存します。プロトコルの各ステップを検証し、パイプラインの頑健性の独立したメトリックを提供する品質管理としてのサンプルと一緒に処理は、明確に定義された細菌モック コミュニティを組み込まれています。スツール、直腸、膣、皮膚、疎性結合、および経口綿棒10,17, を含む他のサンプル タイプに容易に適応可能であるそれとの開始点として使用できるプロトコルとしては説明母乳サンプルに固有ですが、マイクロバイ解析を実行したい研究者。

<table><tbody><tr><td>AllPrep RNA/DNA Mini Kit</td><td>Qiagen</td><td>80204</td><td>DNA/RNA extraction kit</td></tr><tr><td>Eliminase</td><td>Fisher Scientific</td><td>435532</td><td>RNase, DNase, DNA decontaminant</td></tr><tr><td>Thermo Mixer</td><td>Fisher Scientific</td><td></td><td>temperature-controlled vortexer</td></tr><tr><td>Buffer RLT plus</td><td>Qiagen</td><td>1053393</td><td>guanidinium thiocyanate lysis buffer/ Part of Allprep kit</td></tr><tr><td>&amp;szlig;-Mercaptoethanol</td><td>Sigma Aldrich</td><td>63689-25ML-F</td><td>&amp;szlig;-ME is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds</td></tr><tr><td>LME Beads</td><td>MP Biomedicals</td><td>116914050</td><td>bead tube</td></tr><tr><td>QIAgen TissueLyzer</td><td>Qiagen</td><td>85300</td><td>automated sample disruptor adapter set</td></tr><tr><td>QIAshredder column</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>QIAgen RB tube</td><td></td><td></td><td>manufacturer&#039;s microcentrifuge tube in kit</td></tr><tr><td>QIAcube and related plasticware</td><td>Qiagen</td><td>9001292</td><td>automated DNA/RNA purification instrument</td></tr><tr><td>DNA exitus plus</td><td>Applichem</td><td>A7089</td><td>non-enzymatic decontamination solution</td></tr><tr><td>EB Buffer</td><td>Qiagen</td><td>19086</td><td>elution buffer</td></tr><tr><td>QIAgility and related plasticware</td><td>Qiagen</td><td>9001532</td><td>robotic liquid handler</td></tr><tr><td>PCR water</td><td>MO BIO</td><td>17000-</td><td></td></tr><tr><td>5PRIME HotMasterMix</td><td>Quantabio</td><td>2200400</td><td></td></tr><tr><td>Barcoded reverse primers</td><td>Eurofin</td><td>No Catalog #&#039;s</td><td>designed and ordered</td></tr><tr><td>96 well PCR plate</td><td>USA scientific</td><td>1402-9708</td><td></td></tr><tr><td>Tapestation 2200 and related plasticware</td><td>Agilent</td><td>G2964AA</td><td>automated DNA/RNA fragment analyzer</td></tr><tr><td>D1000 reagents for Tapestation</td><td>Agilent</td><td>5067-5585</td><td>Sample buffer and ladder are part of this kit</td></tr><tr><td>OneStep PCR Inhibitor Removal Kit</td><td>Zymo Research</td><td>50444470</td><td>PCR inhibitor removal is done per the manufacturer&#039;s instructions.</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td>DNA clean up kit: silica-membrane-based purification of PCR products</td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher</td><td>Q32854</td><td>dimethylsulfoxide-based dilution buffer and dye are part of this kit.</td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher</td><td>Q33216</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Fisher</td><td></td><td>microvolume spectrophotometer</td></tr><tr><td>MiSeq 300 V2 kit</td><td>Illumina</td><td>15033624/15033626</td><td></td></tr><tr><td>MiSeq</td><td>Illumina</td><td>No Catalog #&#039;s</td><td>next generation sequencer</td></tr></tbody></table>

a method for targeted 16s sequencing of human milk samples

微生物の研究が比較的安価、迅速、かつ高スループット シーケンスの開発と普及します。しかし、これらのすべてのテクノロジと同様再現性のある結果は適切な予防とコントロールを組み込んだ室のワークフローに依存します。これは、バクテリアの DNA を汚染すると誤解を招く結果を生成できます低バイオマス サンプルと特に重要です。この記事は、低半ば throughput スケールの 16S リボソーム RNA (rRNA) V4 領域のターゲット シーケンスを使用して人間の母乳サンプルから微生物を識別するために半自動化されたワークフローを詳しく説明します。プロトコル記述から全乳を含む試料: 換散、核酸抽出、増幅 16S rRNA 遺伝子と品質への取り組みと図書館準備の V4 領域のサンプルします。重要なは、プロトコルおよび議論は顕著な準備と適切な正と負のコントロール、PCR 阻害物質の除去、サンプルの汚染を含む環境、試薬による低バイオマス試料の分析には、問題を考慮または実験のソース、および再現性を確保するために設計されたベスト ・ プラクティスの実験。前述のプロトコルは母乳サンプルに固有ですが、綿棒、きちんとした、または保全バッファーで安定化を凍結で収集されたサンプルを含む多数の低と高バイオマス サンプル タイプに適応されます。

ここで提示されたプロトコルには、生成されたデータのミートがプロトコルの感度、特異性、および汚染制御のベンチマーク テストを確認する重要な品質管理 (QC) 手順が含まれています。プロトコルの最初の品質管理手順に従います PCR 増幅 16S V4 地域 (図 2)。各サンプルからの PCR の製品の 1 つの μ L は 315 450 bp (図 2の赤?...

Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

A Method for Targeted 16S Sequencing of Human Milk Samples

人間のミルク、その他低バイオマス サンプル タイプから 16 s rrna ターゲット シーケンスの半自動化されたワークフローが表示されます。

人間のミルクのサンプルのターゲット 16S シーケンス法

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

a-method-for-targeted-16s-sequencing-of-human-milk-samples

Research

JoVE Journal

Immunology and Infection

9.8K ビュー.  University of California at Los Angeles. 人間のミルク、その他低バイオマス サンプル タイプから 16 s rrna ターゲット シーケンスの半自動化されたワークフローが表示されます。

ビデオ: A Method for Targeted 16S Sequencing of Human Milk Samples

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

水生生物生息地からの炭化水素代謝特性を有する細菌種の単離・増殖・同定

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

環境学

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNAの遺伝子配列決定およびMALDI-TOF MSによるレア細菌性病原体の同定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

免疫・感染学

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

見つけにくい小型哺乳類から高品質のDNAを得るために非侵襲ヘアサンプリング法

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

生物学

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

16SリボソームRNA遺伝子アンプリコンの次世代シーケンシング

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

複雑な動物に関連したサンプルからのシーケンシングメタゲノムとMetatranscriptomes：不純を精製する

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

細菌群集の特徴付けのための効率的な核酸抽出および16S rRNAの遺伝子配列決定

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

マウスの腸内細菌叢に及ぼすバクテリオシンの影響を評価する方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

16S rRNA 増幅配列による糞便の微生物評価ガイドのプロトコル

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Specimen Collection and Analysis of the Duodenal Microbiome

Shifts in the microbiome have been correlated with the physiology and pathophysiology of many organ systems both in humans and in mouse models. The gut microbiome has been typically studied through fecal specimen collections. The ease of obtaining fecal samples has resulted in many studies that have revealed information concerning the distal luminal gastrointestinal tract. However, few studies have addressed the importance of the microbiome in the proximal gut. Given that the duodenum is a major site for digestion and absorption, its microbiome is relevant to nutrition and liver disease and warrants further investigation. Here we detail a novel method for sampling the proximal luminal and mucosal gut microbiome in human subjects undergoing upper endoscopy by obtaining duodenal aspirate and biopsies. Specimen procurement is facile and unaffected by artifacts such as patient preparatory adherence, as might be the case in obtaining colonic samples during colonoscopy. The preliminary results show that the luminal and mucosal microbiomes differ significantly, which is likely related to environmental conditions and barrier functions. Therefore, a combination of duodenal aspirate and biopsies reveal a more comprehensive picture of the microbiome in the duodenum. Biopsies are obtained from the descending and horizontal segments of the duodenum, which are anatomically close to the liver and biliary tree. This is important in studying the role of bile acid biology and the gut-liver axis in liver disease. Biopsies and aspirate can be used for 16S ribosomal RNA sequencing, metabolomics, and other similar applications.

In this manuscript, we discuss a novel method to sample and analyze the duodenal microbiome. This method provides an accurate depiction of microbial diversity and composition in the duodenum and could be useful for further investigation of the duodenal microbiome.

Shifts in the microbiome have been correlated with the physiology and pathophysiology of many organ systems both in humans and in mouse models. The gut ...