Comprehensive Microbiome Profiling Protocol

MO BIO PowerSoil DNA isolation kit provided details about how to manipulate samples; we extracted DNA from soil according to its protocol [51 (link)]. For the stool samples, we extracted metagenomic DNA from approximately 300 mg feces as previously described in the HMP protocol, high temperature was beneficial to the cracking of bacteria, we heated samples at 65 °C for 10 min and 95 °C for 10 min during the operation. Water samples were filtered with sterile 0.22-μm filters, and metagenomic DNA was also extracted from the filter membranes using the MO BIO PowerSoil DNA isolation kit. The DNA was divided into two parts and stored in at − 20 °C until further use. The resulting concentrations and quality of sample DNA were examined via nanodrop instrument and agarose gel electrophoresis.
Based on protocols of NEXTflex Rapid DNA-Seq Kit (Illumina, 96 reactions), some critical process are indispensable to make preparations for sequencing including DNA fragmentation and adapter ligation. We followed the instructions of kit to construct library for sample with the insert size of 350 bp. We lastly sequenced the gut microbiomes of 103 representative samples from 10 bird species and 99 environmental samples (Additional file 10: Table S2). All libraries were then performed sequencing on Illumina Hiseq X10 platform with 2 × 150 bp paired reads (Novogene, Beijing, China).