TABLE 1

Primers used in this study

ApplicationPrimer nameSequence (5′ to 3′)aDegeneracyAnnealing temperature (°C)Amplicon length (bp)Source and/or reference(s)
THDP-PCR for amoA/pmoA gene of comammox, AOB, and MOBA189Y-tagGCCGGAGCTCTGCAGATATCGGNGACTGGGAYTTYTGGb165951327; this study
HD616N-tag (first step)GCCGGAGCTCTGCAGATATCAYCWKVCKNAYRTAYTCNGGb6,144
tag-barcode (second step)XXXXXXXXGCCGGAGCTCTGCAGATATCb,c159
Partial nested PCR for the amoA gene of comammoxA189YGGNGACTGGGAYTTYTGG1652415This study
C576r (first step)GAAGCCCATRTARTCNGCC
CA209fGAYTGGAARGAYCGNCA3250
C576r-barcode (second step)XXXXXXXXXXXXGAAGCCCATRTARTCNGCCc16
PCR for amoA gene of AOBamoA-1F-barcodeXXXXXXXXXXXXGGGGTTTCTACTGGTGGTc15749131
amoA-2RCCCCTCKGSAAAGCCTTCTTC4
PCR for amoA gene of AOAArch-amoAF-barcodeXXXXXXXXXXXXSTAATGGTCTGGCTTAGACGc25363524
Arch-amoARGCGGCCATCCATCTGTATGT1
qPCR for total bacterial 16S rRNA gene338FACTCCTACGGGAGGCAGC15520039, 40
536RGTATTACCGCGGCKGCTG2
qPCR for amoA gene of AOBamoA-1FGGGGTTTCTACTGGTGGT15749131
amoA-2RCCCCTCKGSAAAGCCTTCTTC4
qPCR for amoA gene of comammox clade AA378fTGGTGGTGGTGGTCNAAYTAT855278This study
C616rATCATCCGRATGTACTCHGG6
THDP-PCR for amoAB gene of comammoxA378f-tagGCCGGAGCTCTGCAGATATCTGGTGGTGGTGGTCNAAYTATb850683This study
HDamo/pmoB (first step)GCCGGAGCTCTGCAGATATCCKCATBCKNADRAAYGGYTCb1,152
tag-barcode (second step)XXXXXXXXGCCGGAGCTCTGCAGATATCb,c159
  • a Degenerate 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.

  • b The underlined bases indicate the sequences of the tag.

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