The SD in LH-mcrA amplicon length for one clone in each of the different operational taxonomic units or phylotypes (Fig. 2) ranged from 0.1 to 0.2 bp (Table 1). All partial mcrA gene sequences aligning into the order Birinapant Methanomicrobiales had a 488-bp theoretical amplicon length (from sequencing) but had 483-, 485- or 487-bp phylotypes when experimentally screened by LH-mcrA (Table 1). The majority of the clones related to Methanoculleus had an amplicon length of 483 bp, except phylotypes 7A7 and 7C12 (both at 485-bp). The 7A7 phylotype represented 9% and 5% of the clones in the libraries from PF1 and PF8, respectively. Only one clone

was retrieved

in the libraries that corresponded to the 7C12 phylotype. The clones related to Methanogenium and Methanospirillaceae also had an amplicon length of 485 bp. One clone was related to Methanocorpusculum and had a length of 486.6 bp. Partial mcrA gene sequences aligning within the Methanosarcinaceae family and the Methanobrevibacter spp. had an experimental amplicon length of 481 and 464 bp, respectively. A cluster of unidentified clones (Fig. 2) had amplicon lengths ranging from 466 to 467 bp and were evenly distributed in both PF1 and PF8. Overall, relative abundances using LH-mcrA were in agreement with clone library analyses (Table 1): (1) the 483-bp amplicon accounted for 26% and 70% compared with

33% and 67% of the corresponding clones; (2) the 485-bp amplicon accounted for 40% and 15% compared Bak protein with 34% and 13% of the clones; and (3) the 467-bp amplicon was present at 20% and 13% compared with 19% and 18%; in PF1 and PF8, respectively. One concern with this method is that the variation in amplicon length that distinguishes the Methanomicrobiales and Methanosarcinaceae Phospholipase D1 is only 2 bp (481-, 483- and 485-bp amplicons). Capillary electrophoresis clearly resolved these methanogen groups in mixtures of clones (Supporting Information, Fig. S1 and technical details in Appendix S1). The SD of the amplicon lengths determined on five replicated PCRs ranged between 0.1–0.4 bp (Table S1 in Appendix S2). To test more directly the quantitative aspect of the novel LH-mcrA fingerprint method, PCR products from five different clones having amplicon lengths of 464, 467, 481, 483 or 485 bp were purified and mixed in equal proportion to be used as DNA template in LH-mcrA PCRs. A mean relative abundance and SD of 20.0 ± 3.7% with minimum (for the 483-bp amplicon) and maximum (for the 464-bp amplicon) relative abundances of 13% and 25%, respectively (Table S2 in Appendix S2), were obtained from five LH-mcrA replicated analyses (Table 2, Mixed clones).