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Nā huahana

16S/18S/ITS Amplicon Sequencing-NGS

16S/18S/ITS amplicon sequencing e hōʻike ana i ka phylogeny, taxonomy, a me ka nui o nā ʻano mea i loko o kahi kaiāulu microbial ma o ka noiʻi ʻana i nā huahana PCR o nā hōʻailona genetic housekeeping i loaʻa nā ʻāpana kamaʻilio nui a me nā ʻāpana hypervariable.ʻO ka hoʻokomo ʻana i kēia mau manamana lima molekala maikaʻi e Woeses et al, (1977) e hoʻoikaika i ka hoʻokaʻawale ʻana i ka microbiome profiling.ʻO ke kaʻina o 16S (bacteria), 18S (fungi) a me Internal transcribed spacer (ITS, fungi) hiki ke ʻike i nā ʻano nui ʻelua a me nā ʻano mea ʻike ʻole ʻia.Ua lilo kēia ʻenehana i mea hoʻohana ākea a me ka mea nui i ka ʻike ʻana i ka haku mele microbial ʻokoʻa i nā ʻano like ʻole, e like me ka waha o ke kanaka, nā ʻōpū, nā feces, etc.

Papahana:ʻO Illumina NovaSeq Platform


Nā kikoʻī lawelawe

Nā hopena hōʻike

Nānā Hiʻohiʻona

Pono lawelawe

● ʻO ka hoʻokaʻawale ʻole a me ka ʻike wikiwiki ʻana i ka haku mele microbial i nā laʻana kaiapuni

● Hoʻonā kiʻekiʻe i nā mea haʻahaʻa haʻahaʻa i loko o nā laʻana kaiapuni

● ʻO ke kahe anana QIIME2 hou loa me nā ʻano kālailai like ʻole e pili ana i ka waihona waihona, hōʻike, OTU/ASV.

● High-throughput, ʻoi aku ka pololei

● Pili i nā haʻawina kaiaulu microbial like ʻole

● Loaʻa iā BMK ka ʻike nui me ka ʻoi aku o 100,000 mau laʻana / makahiki, uhi ʻia ka lepo, ka wai, ke kinoea, ka ʻōpala, nā feces, nā ʻōpū, ka ʻili, ka hupa fermentation, nā inike, nā mea kanu, etc.

● Ua maʻalahi ʻo BMKCloud i ka wehewehe ʻikepili i loaʻa iā 45 mau mea hana loiloi pilikino

Nā kikoʻī lawelawe

Ke kaʻina ʻanaPapahana

Hale Waihona Puke

Hāʻawi ʻia ka ʻikepili i manaʻo ʻia

Kuhi ʻia ka manawa huli

ʻO Illumina NovaSeq Platform

PE250

50K/100K/300K mau huaʻōlelo

30 mau lā

Nā loiloi bioinformatics

● Ka hoʻomalu maikaʻi ʻikepili maka

● OTU clustering/De-noise(ASV)

● palapala hōʻike OTU

● ʻokoʻa Alpha

● ʻokoʻa Beta

● Ka nānā ʻana ma waena o ka hui

● Kānāwai hui kūʻē i nā kumu hoʻokolohua

● wānana gene hana

16S

Nā Koina Laʻana a me ka Hoʻouna ʻana

Nā Koina Laʻana:

No ka meaNā unuhi DNA:

ʻAno Laʻana

Ka nui

Hoʻopaʻa ʻana

Maemae

Nā unuhi DNA

> 30 ng

> 1 ng/μl

OD260/280= 1.6-2.5

No nā laʻana kaiapuni:

ʻAno laʻana

Manaʻo ʻia ke kaʻina hana laʻana

lepo

Ka nui o ka laʻana: approx.5 g;Pono e wehe ʻia nā mea maloʻo i koe mai ka ʻili;E wili i nā ʻāpana nui a hele i ka kānana 2 mm;ʻO nā laʻana Aliquot i loko o ka sterile EP-tube a i ʻole cyrotube no ka mālama ʻana.

Feces

Ka nui o ka laʻana: approx.5 g;E hōʻiliʻili a hāʻawi i nā laʻana i loko o ka sterile EP-tube a i ʻole cryotube no ka mālama ʻana.

ʻO loko o ka ʻōpū

Pono e hana ʻia nā laʻana ma lalo o ke kūlana aseptic.Holoi i ka ʻiʻo i hōʻiliʻili ʻia me ka PBS;Centrifuge i ka PBS a ohi i ka precipitant ma EP-tubes.

Sludge

Ka nui o ka laʻana: approx.5 g;E hōʻiliʻili a hāʻawi i kahi laʻana o ka lepo i loko o ka EP-tube sterile a i ʻole cryotube no ka mālama ʻana

Ke kino wai

No ka laʻana me ka liʻiliʻi o ka microbial, e like me ka wai paipu, wai punawai, a me nā mea ʻē aʻe.E mālama i ka membrane i loko o ka paipu sterile.

ʻili

E ʻoki pono i ka ʻili o ka ʻili me ka pulupulu maʻemaʻe a i ʻole ka lāʻau lapaʻau a waiho i loko o ka paipu sterile.

Manaʻo ʻia ka hāʻawi laʻana

E hoʻokuʻu i nā laʻana i loko o ka hau hau no 3-4 mau hola a e mālama i loko o ka wai nitrogen a i ʻole -80 degere a hiki i ka mālama lōʻihi.Pono ka hoʻouna laʻana me ka hau maloʻo.

Holo Hana Hana

hāʻawi laʻana

Hāʻawi laʻana

Hoʻomākaukau hale waihona puke

Kūkulu hale waihona puke

Ke kaʻina ʻana

Ke kaʻina ʻana

ʻIkepili ʻikepili

ʻIkepili ʻikepili

Mahope o ke kuai ana

Nā lawelawe ma hope o ke kūʻai aku


  • Mua:
  • Aʻe:

  • 1. Hoʻolaha ʻano ʻano

    3

    2. Heat palapala 'āina: Species richness clustering

    4

    3. Rare pūʻali puʻupuʻu

    5

    4.NMDS kālailai

    6

    5. Lefse kālailai

    7

     

     

     

    Hihia BMK

    Hōʻike ka poʻe Obese me ka maʻi maʻi type 2 ʻokoʻa a me ka hoʻohui ʻana

    Paʻi ʻia:Pūnaehana Pūnaewele & Microbe, 2019

    Hoʻolālā kaʻina hana:

    Lean non-diabetes (n=633);'Obese non-diabetes (n=494);ʻObese-type 2 diabetes (n=153);
    Māhele ʻāpana: 16S rDNA V1-V2
    Papahana: Illumina Miseq (NGS-based amplicon sequencing)
    Hoʻokomo ʻia ka ʻāpana o nā ʻāpana DNA i ke kaʻina metagenomic ma Illumina Hiseq

    Nā hopena koʻikoʻi

    Ua hoʻokaʻawale maikaʻi ʻia nā hōʻike microbial o kēia mau maʻi metabolic.
    Ma ka hoʻohālikelike ʻana i nā hiʻohiʻona microbial i hana ʻia e 16S sequencing, ua ʻike ʻia ka momona e pili ana i nā loli i ka haku mele microbial, nā hiʻohiʻona pilikino, ʻoi aku ka nui o ka emi ʻana o Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, a pēlā aku. .

    Kuhikuhi

    Thingholm, LB , et al."ʻO ka poʻe obese me ka maʻi maʻi type 2 a me ka ʻole o ka maʻi diabetes e hōʻike i ka mana a me ka hoʻokumu ʻana o ka ʻāʻī microbial."Pūnaehana Pūnaewele & Microbe26.2(2019).

     

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