16S rRNA of contents from feces: patient
Feces (30 mg) mixed with 3.5 mL of Microbial Lysis Buffer 1, 100 L of Proteinase K as well as 30 L of RNase A followed by upside-down mixing multiple times. After incubating at 37C for 30 mins, add 1.2 mL of Microbial Lysis Buffer 2 followed by upside-down mixing. The reaction was incubated at 55C for 120 mins and then centrifuged for 10 mins. Transfered the obtained supernatant and run through Genomic-tip columns to purification genomic DNA. Adding the desired amount of EB buffer into sample tubes depending on the size of precipitated DNA. Incubate at 37C for 30-60 mins until DNA samples are completely dissolved for further nucleic acid quality control step. The qualified library will be sequenced on the PacBio platform. Raw data are filtered to generate high quality clean data through quality control with iTools Fqtools fqcheck (v.0.25), remove adaptor with cutadapt (v.2.6), and filtering with readfq (v1.0). If paired-end reads overlap with each other, then a consensus sequence will be generated by FLASH (Fast Length Adjustment of Short reads, v1.2.18).