Transcription of 16Sサンプル調製ガイド15044223 A JPN - Illumina, Inc.
1 15044223 Rev. B JPN1 16S Metagenomic SequencingLibrary Preparation miseq 16S RNA 216S 5 PCR6 PCR 8 PCR10 PCR 213 15 16 miseq 17 MiSeqReporter 20 21 16S RNA 16 SrRNA 1.
2 500bp 9 16 SrRNA 16 SrRNA 16 SrRNA V3 V4 miseq MiSeqReporter BaseSpaceSequenceHub 16 SrRNA 16S 1 V3-V4 1 460bp 3 2 V3-V4 PCR NexteraXT 96 3 miseq MiSeqv3 300bp V3-V4 65 1 miseq 2.
3 000 96 1 10 4 MSR BaseSpaceSequenceHub MiSeqReporter BaseSpaceSequenceHub Greengenes 16 SrRNA 16 SrRNA GenerateFASTQ MiSeqReporter 20 2 116S V3-V4 DNA PCR v3 MiSeqV3 16SV3 V4 Klindworth KlindworthA,PruesseE,SchweerT,PepllesJ,Q uastC,etal.
4 2013 (1). IUPAC 16S PCR =5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTA CGGGNGGCWGCAG16S PCR =5'GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAC TACHVGGGTATCTAATCC 3 16S 16S 1 5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[ ] 5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[ ] a 50bp 2x300bp 550bp b Tm 60 65 C PCR Tm c 21 4 16S 16S
5 216S 16S 5 PCR PCR DNA 3 21 DNA 10mMTris-HCl 1 L-15 -25 C PCR 1 M 1 5 L-15 -25 C PCR 1 M 1 5 L-15 -25 C2xKAPA HiFiHotStart ReadyMix1 L-15 -25 CMicroseal A 96 mL PCR 1 Agilent DNA1000 5067-1504 1 DNA 2xKAPAHiFiHotStartReadyMix DNA 5 ng/ L L PCR 1 M5 L PCR 1 M5 L2xKAPA HiFiHotStart L 25 L PCR6 2 PCR 95 C3 25 95 C30 55 C30 72 C30 72 C5 4 C 3 BioanalyzerDNA1000 PCR 1 L V3 V4 PCR 550bp 3 PCR PCR7 PCR AMPureXP 16SV3 V4 10 L-15 -25 CAMPureXP 1 20 L2 8 C 80% EtOH 1 400 L96 mL PCR 1 Microseal B 96 MIDI 1 AMPureXPBeads
6 1 PCR 20 C 1,000 g 1 2 - 25 L PCR PCR MIDI 96 MIDI 96 PCR 3 AMPureXP 30 4 20 L AMPureXP PCR 596 PCR 10 MIDI 1,800rpm 2 6 CLP 5 7 CLP 2 8 PCR PCR 8 9 PCR 80% a 80% 200 L b 30 c 10 PCR 2 a 80% 200 L b 30 c d P20 11 PCR 10 12 PCR
7 10mMTris-HCl PCR L 13 10 1,800rpm 2 14 2 15 2 16 PCR 96 PCR 50 L PCR Microseal B -15 -25 C 1 PCR 9 PCR NexteraXTIndexKit 2xKAPA HiFiHotStart ReadyMix1 25 L-15 -25 CNexteraXT IndexKit NexteraXT 1 N7XX FC-131-1001 FC-131-1002 1 5 L-15 -25 CNexteraXT IndexKit FC-131-1001 FC-131-1002 NexteraXT 2 S5XX 1 5 L-15 -25
8 CPCR 1 10 LTruSeq FC-130-1005 196 mL PCR 1 Microseal A 1 1 5 L 96 45 L 2 1 2 TruSeq a 2 A H b 1 1 12 23 PCR10 4 TruSeq A 2 B 1 C96 3 PCR 5 L 96 PCR TruSeq 4 DNA 1 2 2xKAPAHiFiHotStartReadyMix PCR DNA5 LNexteraXT 1 N7xx 5 LNexteraXT 2 S5xx 5 L2xKAPA HiFiHotStart ReadyMix25 LPCR 10 L 50 L5 10 6 Microseal A 720 C 1.
9 000xg 1 PCR11 8 PCR 95 C3 8 95 C30 55 C30 72 C30 72 C5 4 C PCR12 PCR 2 AMPureXP 10 L-15 -25 CAMPureXP 1 56 L2 8 C 80% EtOH 1 400 L96 mL PCR 1 Microseal B 96 MIDI 1 1 PCR 20 C 280 g 1 2 - 50 L PCR PCR MIDI 96 MIDI 96 PCR 3 AMPureXP 30 4 56 L AMPureXP PCR 596 PCR 10 MIDI 1.
10 800rpm 2 6 CLP 5 7 CLP 2 8 PCR PCR 213 9 PCR 80% a 80% 200 L b 30 c 10 PCR 2 a 80% 200 L b 30 c d P20 11 PCR 10 12 PCR 10mMTris-HCl PCR L 1396 PCR 10 MIDI 1,800rpm 2 14
