16S/18S/ITS Amplicon Sequencing-NGS
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
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
Kūkulu hale waihona puke
Ke kaʻina ʻana
ʻIkepili ʻikepili
Nā lawelawe ma hope o ke kūʻai aku
1. Hoʻolaha ʻano ʻano
2. Heat palapala 'āina: Species richness clustering
3. Rare pūʻali puʻupuʻu
4.NMDS kālailai
5. Lefse kālailai
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).
