TABLE 1.

Primers used in this study

Application	Primer name	Sequence (5′ to 3′)a	Degeneracy	Annealing temperature (°C)	Amplicon length (bp)	Source and/or reference(s)
THDP-PCR for amoA/pmoA gene of comammox, AOB, and MOB	A189Y-tag	GCCGGAGCTCTGCAGATATCGGNGACTGGGAYTTYTGGb	16	59	513	27; this study
	HD616N-tag (first step)	GCCGGAGCTCTGCAGATATCAYCWKVCKNAYRTAYTCNGGb	6,144
	tag-barcode (second step)	XXXXXXXXGCCGGAGCTCTGCAGATATCb,c	1	59
Partial nested PCR for the amoA gene of comammox	A189Y	GGNGACTGGGAYTTYTGG	16	52	415	This study
	C576r (first step)	GAAGCCCATRTARTCNGCC
	CA209f	GAYTGGAARGAYCGNCA	32	50
	C576r-barcode (second step)	XXXXXXXXXXXXGAAGCCCATRTARTCNGCCc	16
PCR for amoA gene of AOB	amoA-1F-barcode	XXXXXXXXXXXXGGGGTTTCTACTGGTGGTc	1	57	491	31
	amoA-2R	CCCCTCKGSAAAGCCTTCTTC	4
PCR for amoA gene of AOA	Arch-amoAF-barcode	XXXXXXXXXXXXSTAATGGTCTGGCTTAGACGc	2	53	635	24
	Arch-amoAR	GCGGCCATCCATCTGTATGT	1
qPCR for total bacterial 16S rRNA gene	338F	ACTCCTACGGGAGGCAGC	1	55	200	39, 40
	536R	GTATTACCGCGGCKGCTG	2
qPCR for amoA gene of AOB	amoA-1F	GGGGTTTCTACTGGTGGT	1	57	491	31
	amoA-2R	CCCCTCKGSAAAGCCTTCTTC	4
qPCR for amoA gene of comammox clade A	A378f	TGGTGGTGGTGGTCNAAYTAT	8	55	278	This study
	C616r	ATCATCCGRATGTACTCHGG	6
THDP-PCR for amoAB gene of comammox	A378f-tag	GCCGGAGCTCTGCAGATATCTGGTGGTGGTGGTCNAAYTATb	8	50	683	This study
	HDamo/pmoB (first step)	GCCGGAGCTCTGCAGATATCCKCATBCKNADRAAYGGYTCb	1,152
	tag-barcode (second step)	XXXXXXXXGCCGGAGCTCTGCAGATATCb,c	1	59

^aDegenerate bases: R, A/G; Y, C/T; K, G/T; S, G/C; W, A/T; H, A/T/C; V, A/G/C; B, G/C/T; and N, A/T/C/G.

^bThe underlined bases indicate the sequences of the tag.

^cXXXXXXXX and XXXXXXXXXXXX indicate the presence of 8- and 12-base barcodes, respectively, used to differentiate samples in high-throughput sequencing mixtures.