-DRR011117 454 GS Junior sequencing: HXVJWSB01__Yaku_0782__ITS3 SAMD00008419 rhizosphere metagenome DRA001010 PCR 2015-06-19 163157 23102 LS454 ARCHIVAL_FILE fasp.sra.ebi.ac.uk:/vol1/drr/DRR011/DRR011117 ftp.sra.ebi.ac.uk/vol1/fastq/DRR011/DRR011117/DRR011117.fastq.gz 1352764800000 Generic HXVJWSB01__Yaku_0782__ITS3 ftp.sra.ebi.ac.uk/vol1/drr/DRR011/DRR011117 b737064403c17493036beee0987a0556 DRP001052 104 Complex community structure of ectomycorrhizal, arbuscular-mycorrhizal and root-endophytic fungi in a mixed subtropical forest of ectomycorrhizal and arbuscular-mycorrhizal plants GENERATED_FILE HXVJWSB01__Yaku_0782__ITS3 454 GS Junior 454 GS Junior sequencing: HXVJWSB01__Yaku_0782__ITS3 105143a627249be0bcc20c7755c1adcf ftp.sra.ebi.ac.uk/vol1/fastq/DRR011/DRR011117/DRR011117.fastq.gz 51498 AMPLICON METAGENOMIC ftp.sra.ebi.ac.uk/vol1/drr/DRR011/DRR011117 SINGLE dcc_metagenome public fasp.sra.ebi.ac.uk:/vol1/fastq/DRR011/DRR011117/DRR011117.fastq.gz 1;2787823;12908;408169;410657;939928 SAMD00008419 DRR011117 We sequenced fungal ITS sequences based on a tag-encoded massively-parallel pyrosequencing. For each root sample, the entire ITS region and partial ribosomal large subunit region was amplified using the fungus-specific high-coverage primer ITS1F_KYO2 and the universal primer LR3 (http://www.biology.duke.edu /fungi/mycolab/primers.htm). PCR was conducted using a temperature profile of 95??C for 10 min, followed by 20 cycles at 94??C for 20 s, 50??C for 30 s, 72??C for 120 s and a final extension at 72??C for 7 min with a buffer system of Ampdirect Plus and BIOTAQ HS DNA Polymerase (Shimadzu, Kyoto, Japan). The PCR product of each root sample was subjected to a second PCR step targeting the ITS2 region. The second PCR was conducted with a universal primer ITS3_KYO2 fused with the 454 Adaptor A and each sample-specific molecular ID, and the reverse universal primer LR_KYO1b (5'-MGC WGC ATT CCC AAA CWA-3') fused with the 454 Adaptor B. A buffer system of Taq DNA Polymerase with Standard Taq Buffer (New England BioLabs, Ipswich, MA, USA) was used under a temperature profile of 95??C for 1 min, followed by 40 cycles at 94??C for 20 s, 50??C for 30 s, 72??C for 60 s and a final extension at 72??C for 7 min. The ITS amplicons of the second PCR step were subjected to pyrosequencing. The first 576 and the second 624 samples were sequenced separately using a GS Junior sequencer (Roche). The rbcL amplicons of the first 480 root samples were pooled and purified using ExoSAP-IT (GE Healthcare) and QIAquick PCR Purification Kit (QIAGEN). The sequencing of the first 576 samples was conducted as instructed by the manufacturer. Likewise, the amplicons of the remaining 624 samples were pooled and purified, and then sequenced in the second run. DRX010073 KYOTO_HE HXVJWSB01__Yaku_0782__ITS3 2014-04-28 DRX010073 rhizosphere metagenome 939928 PRJDB2078 DRR011117 DRS009918 2014-04-28 PRJDB2078
0 commit comments