mcrA gene amplicon classification and distribution across lake sediment samples

Amplicon libraries were prepared as described in Han et al. 2020 (https://doi.org/10.1111/1462-2920.15115) and involved the same mcrA primer combination as for qPCR. Sequencing was done on an Illumina MiSeq (Illumina Inc.). Raw sequence reads were quality-checked by FastQC, read ends trimmed using seqtk, paired end reads merged into amplicons by FLASH, primer sites trimmed by usearch, and quality filtering was done by prinseq. Zero-radius Operational Taxonomic Units (ZOTUs) were generated using UNOISE3 and clustered using a 97% similarity threshold to generate OTUs. OTUs were taxonomically identified based on neighbor-joining phylogenetic trees in ARB. This mcrA database used for classification contained >2,400 high-quality mcrA sequences from a wide range of published amplicon and whole-genome sequencing studies with manually optimized sequence alignments (https://doi.org/10.3389/fmicb.2023.1192029).