Pyrosequencing analyisis on bacteria within permafrost soils along CRCOP
Soil DNA were extracted from the upper and bottom parts in the active layer and the permafrost table as well. Each horizons took three replicates. To investigate the response of indigenous bacterial community to crude oil contamination, a 8-weeks simulated contamination with crude oil which is obtained from the pipeline was conducted. The soil samples were contaminated with 30% and 50% (v/w) crude oil for active layer soils and 30% for deep active layer and upper permafrost table. Pooled DNA were PCR amplified with bacterial universal primer set 8F/533R with specially-designed barcodes.After purification with MiniElute PCR purification kit (Qiagen), parallel tagged sequencing was performed using a Roche 454 GS FLX in Majorbio. Barcode was used to label different samples. The information is as follows:    forward TTACCGCGGCTGCTGGCAC  barcode AGCTAGATAC      1300 (sample label,the same below)   barcode AGCTGTCGAC      1330  barcode AGTATGCACG      1350  barcode AGTCGCGCTA      1500  barcode AGTCTGTCTG      1530  barcode ATACACACGA      1600  barcode ATACGCGTGC      1630  barcode ATACTAGCAC      2300  barcode ATAGAGCTAG      2330  barcode ATATAGAGTA      2350  barcode ATCGCTCACG      2500  barcode ATCGTCAGTC      2530  barcode ATCTCTCGTA      2600  barcode ATCTGAGACG      2630  barcode ATGCTACGTC      3300  barcode ATGTGACTAC      3330  barcode CACGAGACAG      3350  barcode CACGCGAGTC      3500  barcode CACGCTACGA      3530  barcode CACGTGTATA      3600  barcode CACTACGATG      3630  barcode CACTATACTC      4300  barcode CAGCGTACTG      4330  barcode CAGTCTCTAG      4350  barcode CATAGTCGCG      4500  barcode CGAGACACTA      4530  barcode CGAGAGTGTG      4600  barcode CGAGTCATCG      4630