Laboratory protocol: UMI labelling and amplicon multiplexing
We developed a procedure (MAUI-seq) to amplify multiple target genes from environmental samples, while assigning a random UMI to each initial copy of a template. We opted for a straightforward protocol using a “one-pot” initiation and amplification system. Forward primers consist of two modules; an inner primer bearing the UMI and designed to amplify the target gene, and a universal outer primer that binds only to a linker on the inner primer (Figure 1A ). We used a 12-base UMI that allowed over 4 million distinct sequences, which is adequate to ensure that duplicate use is negligible for samples with a few thousand sequenced UMIs. For studies with greater sequencing depth, a longer UMI can easily be designed. As a test case, we used MAUI-seq to investigate the genetic diversity of the nitrogen-fixing bacterium Rhizobium leguminosarum symbiovar trifolii (Rlt) by characterising amplicons from the chromosomal core genes rpoB and recAand the plasmid-borne nodulation genes nodA and nodD . Each gene was amplified separately in a single reaction, using a target-specific inner forward primer (at low concentration) to assign the UMI and a universal outer primer (at high concentration) to amplify the resulting molecules (Figure 1A ). The resulting amplicons were pooled and tagged by Nextera to identify the sample, then further pooled for high-throughput paired-end sequencing (Figure 1B ). The full MAUI-seq step-by-step laboratory protocol can be found inAdditional File 1 .