粪便样本不同保存方式对肠道菌群测序结果的影响
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国家重点研发计划(2018YFC1603803,2018YFC2000505);国家自然科学基金(81991534)


Effects of different preservation methods for fecal samples on the sequencing results of gut microbiota
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    摘要:

    【目的】本文探究了3种室温保存剂和–80℃冷冻保存对粪便样本中菌群结构的影响,为大规模、标准化的采样提供参考。【方法】本研究采集了5名健康志愿者的新鲜粪便作为测试样本,采用4种不同的保存方式保存:DETs室温保存、GITC室温保存、RNAlater室温保存和–80℃冷冻保存,在保存0、1、3、7、14、28 d后,采用高通量测序技术测定样本中微生物16S rRNA基因V3-V4区序列,通过比较不同保存剂、不同保存时间后的样本与新鲜样本的差异,分析保存剂和保存时间对粪便样本菌群组成及丰度的影响。【结果】5组样本共得到489个操作分类单元(operational taxonomic units,OTUs),其中488个OTUs为5组所共有,且样本alpha多样性无统计学差异。从属水平上看,与fresh组(新鲜样本)相比,DETs组的拟杆菌属相对丰度显著升高,且部分OTUs在保存超过3 d后发生明显变化;其余保存方式无显著差异。综合分析微生物群落多样性和丰度,结果表明,在保存不同天数后,4种保存方式的样本与fresh组的菌群结构均无显著差异,其中-80℃保存的样本与fresh组样本菌群结构相似程度最高,但不同志愿者与各自新鲜样本的相似程度变化较大,且整体相似程度随着时间的推移与fresh组差异呈下降趋势;而使用DTEs保存剂和GITC保存剂保存的样本,志愿者个体间差异小,保存效果稳定,且随时间变化小。聚类分析结果表明,无论使用哪种保存方式,保存方式和时间带来的差异均小于志愿者个体间的差异。【结论】在满足低温冷冻保存条件时优先选择–80℃冷冻保存粪便样本,无法满足立即冷冻保存的条件时,可以选择添加保存剂室温保存,且GITC保存剂优于其他保存剂。

    Abstract:

    [Objective] In this study, we compared three room-temperature preservation methods with −80℃ preservation and investigated the effects of these methods on the sequencing results of microbiota in the fecal samples, thence providing guidelines for large-scale and standardized sampling. [Methods] Fresh fecal samples from five healthy volunteers were collected and subjected to four different preservation methods:room-temperature preservation methods (DETs, GITC, and RNAlater) and storage at −80℃ without stabilizers. High-throughput sequencing was deployed to sequence the V3−V4 region of the 16S rRNA genes in the samples being stored for 0, 1, 3, 7, 14 and 28 days. The effects of preservation stabilizers and time on the microbiota composition and species diversity were determined by comparison between different treatments.[Results] A total of 489 operational taxonomic units (OTUs) were obtained from the five groups of samples (fresh samples, three room-temperature samples, and −80℃ preserved samples), among which 488 OTUs were shared by these five groups. No significant difference was observed in the alpha diversity of microbiota amory preservation methods. The relative abundance of Bacteroides in the samples preserved with DETs was higher than that in fresh samples, and some other OTUs in the samples preserved with DETs changed considerably after 3-day storage. No significant difference in the relative abundance of microbial genera was observed amory other groups apart from the DETs group. The microbiota diversity and abundance showed no significant difference between the samples preserved with the four methods and the fresh samples after the storage for different days. The samples stored at −80℃ had the highest similarity to the fresh samples. However, the similarity of the samples from each volunteer varied, and such variation tended to increase with the prolonging of storage time. Nevertheless, DTEs and GITC methods exhibited outstanding stability, especially after prolonged storage time. Cluster analysis revealed that the variation associated with preservation methods and time was neglectable. [Conclusion] Subject to the availability, storage at −80℃ is considered the gold standard for fecal samples, and storage at room temperature with the addition of GITC shows comparable performance to low temperature preservation.

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    [17] Song SJ, Amir A, Metcalf JL, Amato KR, Xu ZZ, Humphrey G, Knight R. Pres汥汲慶牡摴?坯????牴敨捯此?匠??剦潦摥牲椠杩畮攠穦?????敭湩獣浲潯牢敩?????坴敡獢瑩敬物??????慥正整物?剧????癴慡汢畩慬瑩楴潹渠?潯晲?晦敩捥慬汤??乴??灩牥敳献攠爼癩愾瑭楓潹湳?瑥敭捳格港楩焾甬攠猲‰愱渶搬?攱昨昳攩挺瑥猰‰漰昲?献愼浢灲氾敛?愸杝攠?慡湮搠?搬椠效瑡?漠湌?朠敌湩潮琠祙瀬椠湌杩?獆甬挠捗敡獮獧???椠?呡桮敧??漬甠牘湩慡汯?潌昬?坋楲汩摳汴楩晡敮??慮渠態本攠浊敩湡琠??椠?????ㄠ????????????????????扥牡?孥??崠?乯楲猠桲楯浯潭琠潴?奭???楡穴畵瑲慥渠楳?卯??乧慥欠慡橮楤洠慴?呡???潯獲潴搠慯????坣慡瑬愠湳慡扭数????卦慯楲琠潭?奴??卥桮楯扭慩瑣愠?呮??她慳捥桳椮搠愼?匾??奣慲浯慢摩慯?呥???椾本栠′猰琱愸戬椠氶椨琱礩?漴昳?昼慢敲挾慛氱?浝椠掁犛漬戠楎濻洬攠″捑漬洠灵濋獳椬琠榞漏滕?椠渤?枪疿懝湘榹擕槹溠敓?璤棓榄演捶禄慱滍愮琠攭?玮漟氁畦瑂槗漬渠′愰琰?爬漠漨洳?琺攲洲瀳攭爲愲琵甮爠教?慡湮搠?爬漠扌畩猠瑍測攠獓獨?摮甠牊椬渠杚?捡潯氠潌湐漬猠捐潡灮祧???椮??畯瑭??楲??????????????????????????扯牬?孳??嵰??漠獰潲浥楳????佴桩湯潮?????甠物慮欠慧浵楴????乲慯瑢獩畯浴敡??楮瑡慬瑹慳湩楳?夠??吾慃湨楩獮慥睳慥?????楮牡慬琠慯?匠??卣畲穯略正楯????丼愯杩愾琬愠欲攰‰吹??丨椳猩栺椲渲漳?吲?‵?椠稨畩杮甠捃桨楩?????椼祢慲挾桛椲‰????畵湨楹猠慆眬愠?????攠瑊栬漠摒?晡漠牍?瀬爠敏瀧慓牵楬湬杩??乮??昬爠潒浯?晳攠捒敐猬?楏渧?条畬慬湡楧摨楡湮攠?琬栠楐潬捡祮慴渠慂瑊攬?獓潴污畮瑴楯潮渠?愮映晔敨捥琠獥???獣?牳删乯??扦慲獥敥摺?灮牧漠景楮氠楦湡来?潡晬?桭畩浣慲湯?浩楯捴牡漠扡楳漠瑤慥?摥楲癭敩牮獥楤琠祵???楧?卭捩楳敥湱琠楳晥楱捵?剮散灩潮牧琠獡??椠????ふ???????????????扩牧?孴??嵮?央愠渼杩 ̄????漠畏????婩栾愬渠朲‰???传由礰愨渳朩?????椹渳‵???塢畲 ̄卛???攠?????慡湮杮?????桨敥湮?兊??坁畭????夬愠湓?????椠慁湢?奥???楃愬渠李?呬???攠????坮慩湧杨?????畃?奩??塎椬愠潓?????桒攮渠?婯??偡牲敩獳敯牮瘠慯瑦椠潣湯?潬晥?瑴桩敯?映敭捥慴汨?獤慳洠灦汯敲猠?慥瑣?慬洠扳楡敭湰瑬?瑳攠浩灮攠牭慩瑣畲牯敢?景潭牥?浳楴捵牤潩扥楳漮琠愼?愾湁慭汥祲獩楣獡?眠楊瑯桵?慮?捬漠獯瑦?故晰晩敤捥瑭楩癯敬?慧湹搼?物放氬椠愲戰氱攷?猠琱愸戵椨氲椩稺攱爱‵攭昱昲挳朮甼瑢? ̄?椲?卝挠楗敨湩捴敥?潂晎?琠桂敯?呧漠瑐慔氬??湥癵楴物潮渠浇攮渠瑐??楳????ぴ?は????ㄠ???ち?㈠???扯牤?孡?つ崠??畳歳畵摥愠?????略橳椠瑦慯?夠???攠瑡敮牡浬楹湳慥瑳椮漠渼?漾晃?瑮桡敤?摡楮猠捊牯極浲楮湡慬渠瑯?猠捚潯牯敬?潧晹?椯湩琾攬猠琱椹渹愱氬?洶椹挨爱漩戺椸漲琭愹‰愮猼?慲 ̄扛椲漳浝愠牆歲敡牮?潺晥?搠楍獁攬愠獓敩?慫挠瑊楂瘬椠瑆祥?楧湵?灯慮琠楊敗測琠獗?睹楮瑥栠?畋氬挠敋牯慨瑮椠癍效?挠潅汭楰瑩楲獩???椠??????慴獩瑯牮漠敯湦琠数牲潥汳??楶????のㄠ??????????扲爠?孡??嵡?匠桄楎潁種愠欼楩 ̄???奥潣湵敬摡慲?卅??奬潯湧敹搼愯?举??失漹渹攸稬愠眷愨?到???愴琲猳甭戱愴礲愸献格楢?吾?′匴敝漠????卨慭楡瑮漠?匬???湮瑧攠獋琬椠湁慲汣?浩楥挠牅潁戬椠潂瑡慲?楥獩?摯椠晌時攬爠敊湯瑨?楳湯?眠潚浐攬渠?睩楬瑳桯?瀠牍故琬攠牋浯?扮椠牊琬栠?牵敡獮甠汍瑌猬?晇牥潳浱?瑩敥牲浥椠湌愬氠?牲敩獥瑮牥楩捳瑥楮漠湌?昬爠慔杵浮敧渠瑊?氠敃湯杭瑭桯?瀠潭汥祴浨潯牤灳栠楦獯浲?慦湥慣污祬猠楳獡???楥?偳?潯卲?佧乥???椠????つㄠ?????????敩????????扩牳?孥??崠??楧獮慡摴慵?呥???湦搠潩桮?????畵牡楬欠楩?????湴瑹攠物??慭湩摣?楯湢瑩牯慭?椠湳摥楱癵楥摮畣慩汮?瘠慤牡楴慡琮椠漼湩猾?楣湩?獮整慩獦潩湣愠汒?慰湯摲?摳愼椯汩社?猠琲愰戱椶氬椠琶椺攳猱‵漱昹?琼桢敲 ̄桛甲洵慝渠?柍申琬?洠榎捉爬漠扎槪漺琬愠?楐湎??愘灦懯渮攠獄故???榲??犠捓梮??楄捎牁澄拝楘潈沜??榰???学て????????????????????戭爲?嬸??崠??慩朠潃捙?吠??卡慮汧稠扙敇爬朠?卩????氠慙獥桮?晚慈猬琠?汩敵渠杘瑄栮?慅摦橦略獣瑴浩敶湥瑮?潳晳?獯桦漠牄瑅?牳攠慢摵獦?瑥潲?楦浯灲爠潩癮整?杳整湩潮浡敬?慭獩獣敲浯扢汥楳攠獄???楰??楳潥楲湶晡潴物浯慮琮椠挼獩??楲????び?ㄠ??㈠??????㈠??????????扥爼?孩??崠′?漰氹本攠爹??????漴核猴攭′???售猠愨摩敮氠????呥牳楥洩洼潢浲愾瑛椲挶?愠?晡汮敡硳楣扩氠敍?琠牂楡洀洀攀爀?昀漀爀?椀氀氀甀洀椀渀愀?猀攀焀甀攀渀挀攀?搀愀琀愀???椀??椀漀椀渀昀漀爀洀愀琀椀挀猀??椀???? ????? ????????????? ??戀爀?嬀??崀??愀瀀漀爀愀猀漀??????甀挀稀礀渀猀欀椀????匀琀漀洀戀愀甀最栀?????椀琀琀椀渀最攀爀?????甀猀栀洀愀渀??????漀猀琀攀氀氀漀??????椀攀爀攀爀?一??倀攀渀愀??????漀漀搀爀椀挀栀??????漀爀搀漀渀??????甀琀琀氀攀礀??????攀氀氀攀礀?匀吀???渀椀最栀琀猀?????漀攀渀椀最??????攀礀?刀????漀稀甀瀀漀渀攀??????挀?漀渀愀氀搀?????甀攀最最攀?????倀椀爀爀甀渀最????刀攀攀搀攀爀????匀攀瘀椀渀猀欀礀??刀??吀甀爀渀戀愀甀最栀?倀???圀愀氀琀攀爀猀?圀???圀椀搀洀愀渀渀????夀愀琀猀甀渀攀渀欀漀?吀??娀愀渀攀瘀攀氀搀?????渀椀最栀琀?刀??儀椀椀洀攀?愀氀氀漀眀猀?愀渀愀氀礀猀椀猀?漀昀?栀椀最栀?琀栀爀漀甀最栀瀀甀琀?挀漀洀洀甀渀椀琀礀?猀攀焀甀攀渀挀椀渀最?搀愀琀愀???椀?一愀琀甀爀攀??攀琀栀漀搀猀??椀???? ? ????????????????戀爀?嬀??崀??漀稀甀瀀漀渀攀?????渀椀最栀琀?刀??唀渀椀昀爀愀挀?愀?渀攀眀?瀀栀礀氀漀最攀渀攀琀椀挀?洀攀琀栀漀搀?昀漀爀?挀漀洀瀀愀爀椀渀最?洀椀挀爀漀戀椀愀氀?挀漀洀洀甀渀椀琀椀攀猀???椀??瀀瀀氀椀攀搀??椀???椀?愀渀搀??渀瘀椀爀漀渀洀攀渀琀愀氀??椀挀爀漀戀椀漀氀漀最礀??椀????  ?????????????????????戀爀?嬀??崀??愀氀愀渀欀愀????匀愀氀漀渀攀渀????匀愀氀漀樀愀爀瘀椀????刀椀琀愀爀椀?????洀洀漀渀攀渀?伀???愀爀挀椀愀渀椀?????漀眀氀愀渀搀?倀???漀愀搀?????愀爀猀攀搀?????愀洀????倀愀氀瘀愀????匀瀀椀氀氀攀爀?刀???搀攀?嘀漀猀?圀????昀昀攀挀琀猀?漀昀?戀漀眀攀氀?挀氀攀愀渀猀椀渀最?漀渀?琀栀攀?椀渀琀攀猀琀椀渀愀氀?洀椀挀爀漀戀椀漀琀愀???椀??甀琀??椀???? ???????? ?????????????戀爀?嬀??崀??漀渀最洀椀爀攀??????愀氀琀戀椀攀?????愀欀攀爀?刀???唀猀攀?漀昀??氀礀猀椀猀?戀甀昀昀攀爀??椀渀??一??椀猀漀氀愀琀椀漀渀?愀渀搀?椀琀猀?椀洀瀀氀椀挀愀琀椀漀渀?昀漀爀?洀甀猀攀甀洀?挀漀氀氀攀挀琀椀漀渀猀????????戀爀?嬀??崀?一攀挀栀瘀愀琀愀氀?????刀愀洀??????愀猀猀漀渀?????一愀洀瀀爀愀挀栀愀渀?倀??一椀攀挀?匀刀???愀搀猀栀愀??娀???愀琀栀攀爀氀礀??????愀樀甀洀搀愀爀??倀???愀琀漀?????攀挀愀氀?挀漀氀氀攀挀琀椀漀渀??愀洀戀椀攀渀琀?瀀爀攀猀攀爀瘀愀琀椀漀渀??愀渀搀??一??攀砀琀爀愀挀琀椀漀渀?昀漀爀?倀?刀?愀洀瀀氀椀昀椀挀愀琀椀漀渀?漀昀?戀愀挀琀攀爀椀愀氀?愀渀搀?栀甀洀愀渀?洀愀爀欀攀爀猀?昀爀漀洀?栀甀洀愀渀?昀攀挀攀猀???椀??漀甀爀渀愀氀?漀昀??椀挀爀漀戀椀漀氀漀最椀挀愀氀??攀琀栀漀搀猀??椀????  ?????????????????
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范馨月,陈璐萍,朱金滔,律娜,朱宝利. 粪便样本不同保存方式对肠道菌群测序结果的影响[J]. 微生物学报, 2022, 62(2): 520-532

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