TABLE 1.

Names, target groups, nucleotide sequences, primer lengths, amplicon lengths, annealing temperatures, and amplification success of mcrA and archaeal 16S rRNA gene primers with DNA extracts from Everest Mound^a

Name	Target groups	Primer sequences	No. of nucleotides	Length (bp)	Tanneal (°C)	Amplificationb	Specificityc
mcrA gene primers
mcrI	General	F, 5′-TAYGAYCARATHTGGYT	17; 17	∼490	51	+	+
		R, 5′-ACRTTCATNGCRTARTT
ME1/ME2	General	F, 5′-CMATGCARATHGGWATGTC	20; 21	∼750	58	+	+
		R, 5′-TCATKGCRTAGTTDGGRTAGT
mcrIRD	General except ANME-1	F, 5′-TWYGACCARATMTGGYT	17; 17	∼490	55	+	+
		R, 5′-ACRTTCATBGCRTARTT
ANME-1-mcrI	ANME-1, ANME-1-related group	F, 5′-GACCAGTTGTGGTTCGGAAC	20; 20	∼480	63	+	+
		R, 5′-ATCTCGAATGGCATTCCCTC
mcrMS	Methanosarcina	F, 5′-GACCAGATCTGGCTCGGATC	20; 20	425	66	+	−
		R, 5′-TCGCCCTGGTAGGACAGAAC
mcrANME-2	ANME-2	F, 5′-GGATTCACGCAGTACGCAAC	20; 20	155	64	+	+
		R, 5′-CAAGAAGCGTTGGGTAGTCC
mcrMsaeta	Methanosaeta	F, 5′-TACACCAACGATGTCCTGGA	20; 20	305	64	+	−
		R, 5′-CACTGATCCTGCAGGTCGTA
mcrURFS	Unidentified Rice Field Soil McrA/Zoige cluster	F, 5′-TATGCAACACCAGCATACACC/GTATGCCACAGCAGCATACAC	22/21; 17	385	64	+	−
		R, 5′-CACCGCACTGATCCTGC
mcrAM-3 I	ANME-3	F, 5′-GATATCATTCAGACAAGCCG	20; 20	525	60	−	NA
		R, 5′-AGTTCAAGAGGCTCTCCTTC
mcrAM-3 II	ANME-3	F, 5′-CCTTGAGGTAGTCGGTGCAG	20; 22	480	64	−	NA
		R, 5′-AGTTCAAGAGGCTCTCCTTCGT
mcrAM-3etal	ANME-3, Methanococcoides, Methanomethylovorans, Methanolobus, Methanohalophilus	F, 5′-GATATCATTCAGACAAGCCGTR, 5′-CACCACACTGGTCCTGC	21; 17	480	60	+	−
mcrRCI/FC	Rice Cluster I, Fen cluster	F, 5′-TACAAGATGTGCGCCGGT	18; 20	560	64	−	NA
		R, 5′-CATGCTTCCTTGTGCAGGTA
mcrFCI	Fen cluster	F, 5′-AGCCAGGTGGCATCAAGTT	19; 20	510	64	−	NA
		R, 5′-ACTGGTCCTGCAGGTCGTAG
mcrFCII	Fen cluster	F, 5′-AGCCAGGTGGCATCAAGTT	19; 19	445	64	+	−
		R, 5′-GACAGGTACCAGCCGTTCA
mcrMcorp	Methanocorpusculum	F, 5′-TGTCATCAACATGGCCCAC	19; 20	550	64	+	−
		R, 5′-TCGTAGCCGAAGAAACCAAG
mcrMspir	Methanospirillum	F, 5′-GATGAGTTCACCTACTATGGTATR, 5′-CTGACAGAGAGTGAGTTGGT	23; 20	335	56	+	−
mcrMmicrob	Methanomicrobiales	F, 5′-CACCTACTACGGTATGGACTA	21; 20	310	61	+	(−/+)
		R, 5′-GAGTTTGCTGAACCACACTG
mcrMcul	Methanoculleus and close relatives	F, 5′-GGTATGGACTACATCAAGGACAAR, 5′-ACTGGTCCTGGAGGTCGTA	23; 19	290	62	+	(−/+)
mcrGMcul	Guaymas Basin Methanomicrobiales mcrA cluster	F, 5′-CACCTACTACGGTATGGACTA	21; 18	355	61	+	+
		R, 5′-AGCTCTCCGAGCAGACCT
mcrMbacA	Methanobacterium aarhusense group	F, 5′-GCAAAACACGCAGAAGTTGT	20; 22	515	63	+	−
		R, 5′-GTCTGGAGTGCTGTTCTTTGTG
mcrMbac	Methanobacteriales except M. aarhusense and Methanothermobacter	F, 5′-GGTTAGGTTCTTACATGTCTGGTGR, 5′-GCACCACATTGATCTTGTAAATC/TGCTCCACACTGGTCCTG/CACCACACTGGTCCTGGA	24; 23	365	63	+	(−/+)
mcrMcoc	Methanococcaceae	F, 5′-AAGAAGAGCAAGAGGTCCAAA	21; 23	490	61	+	−/+
		R, 5′-TCGTATCCGTAGAATCCTAATCT
mcrMtb	Methanothermobacter	F, 5′-AGCCTACACAGACAACATCCTCR, 5′-CACCACACTGGTCCTGGA	22; 18	315	63	+	−
mcrMpK	Methanopyrus kandleri	F, 5′-CTAGGATCCTACATGTCAGGAGGR, 5′-CCTCACGCTCAGCGAGTT	23; 18	385	64	+	+
mcrDBGrII	Deeply Branching mcrA group II	F, 5′-GGGAGTAGGATTCACGCAGTA	21; 22	410	64	+	+
		R, 5′-GATAGTTTGGACCACGCAGTTC
mcrMlas	Clone mlas, clone DEBITS and relatives	F, 5′-ACGACTTCTGCTACTACGGTGCR, 5′-CCTGCCCATCTCCTCCTT	22; 18	255	64	+	−
mcrDBGrIII	Deeply Branching mcrA group III	F, 5′-GCAGTATGCAACCGCTGTT	18; 19	395	64	+	+
		R, 5′-GTCTGCACCTCTGAGCTCAAG
mcrMpyralesI&II	Methanopyrus mcrA subclusters I and II	F, 5′-GTGTACACGGACAACATCCTGGR, 5′-ACGCTCAGCGAGTTGGC	22; 17	330	64	+	−
Archaeal 16S rRNA gene primers
MS 183F/MS 1138R	Methanosarcina	F, 5′-TGCTGGAATGCTTTATGCGT	20; 19	955	65	−	−
		R, 5′-CCGGAGGACATGCTGGTAA
MS 184F/MS 1009R	Methanosarcina	F, 5′-GCTGGAATGCTTTATGCGT	19; 20	825	62	+	−
		R, 5′-TGGCCTACATATTGCTGTCG
MCC 221F/MCC&Mlob 1155R	Methanococcoides, Methanolobales, Methanohalophilus	F, 5′-CCTAAGGATGGATCTGCGG	19; 21	934	64	+	−/+
		R, 5′-CCACAGAGTACCCATCATCCC
RCI 549F/RCI 1014R	Rice Cluster I	F, 5′-TGGTGGCCGATATTATTGAGTC	22; 20	465	64	+	(−/+)
		R, 5′-TCAGCCTGGCCTTCATACAA
ANME-2 244F/ANME-2 836R	ANME-2	F, 5′-TCAGGTTGTAGTGGGTGTAA	20; 20	592	60	−	NA
		R, 5′-CTGACACATAGCGAGCATCG
Msta 268F/Msta 927R	Methanosaeta	F, 5′-CCTACTAGCCTACGACGGGT	20; 21	659	62	+	(−/+)
		R, 5′-CCCGCCAATTCCTTTAAGTTT
Msta 571F/Msta 927R	Methanosaeta	F, 5′-TAAAGGGTCTGTAGCCGGCC	20; 21	356	65	+	(−/+)
		R, 5′-CCCGCCAATTCCTTTAAGTTT
MM 48F/MM 1171R	Methanomicrobiales	F, 5′-TTAAGCCATGCGAGTCGAGA	20; 19	1123	65	+	+
		R, 5′-TTTAGCAGAGGCGGTCCCA
ANME-1 42F/ANME-1 898R	ANME-1	F, 5′-GAGTTCGATTAAGCCATGTTAGTR, 5′-CGACCGTACTCCCCAGAT	23; 18	856	60	+	+
ANME-1 Deep 35F/ANME-1 Deep 1038R	Deeply Branching ANME-1	F, 5′-GCTATCAGCGTCCGACTAAGC	21; 19	1003	65	+	(−/+)
		R, 5′-TAATCCGGCAGGGTCTTCA
ANME-1 Deep 176F/ANME-1 Deep 1038R	Deeply Branching ANME-1	F, 5′-CGGATAGGCCTCTGATACCTG	21; 19	852	64	−	NA
		R, 5′-TAATCCGGCAGGGTCTTCA
MB 136F/MB 873R	Methanobacteriales	F, 5′-CCTTAGGACTGGGATAACCC	20; 19	737	62	−	NA
		R, 5′-TTAACAGCTTCCCTTCGGC
MB 137F/MB 873R	Methanobacteriales	F, 5′-CTTGGGACCGGGATAACC	18; 19	736	64	−	NA
		R, 5′-TTAACAGCTTCCCTTCGGC
MB 310F/MB 873R	Methanobacteriales	F, 5′-CGGAGATGGAACCTGAGACA	20; 19	563	64	−	NA
		R, 5′-TTAACAGCTTCCCTTCGGC
MC 266F/MC 910R	Methanococcales	F, 5′-GCCCACCAAGCCTACGATC	19; 22	644	65	−	NA
		R, 5′-TTTCAGTCTTGCGACCGTACTC
MP&MT 235F/	Methanopyrales and Methanothermales	F, 5′-TGCGGCCGATTAGGTAGTT	19; 20	786	64	+	(−/+)
MT 1021R		R, 5′-AAGGTCATCAACCTGGCCAT
MP&MT 235F/	Methanopyrales	F, 5′-TGCGGCCGATTAGGTAGTT	19; 20	748	64	+	(−/+)
MP 983 R		R, 5′-TAAGGTTTCCGGCGTTGAAT

^aWith the exception of the mcrI and ME1/ME2 primers, which were designed by Springer et al. (13) and Hales et al. (16), all primers are from Lever (15).

^b+, PCR amplification occurred; −, no PCR amplification occurred.

^c+, mcrA genes of target group only were amplified; −/+, mcrA genes of target group and others were amplified; (−/+), mcrA genes of nontarget groups were amplified; −, only non-mcrA genes were amplified. NA, not applicable.