Table 1.
Primer sequences used for amplification of the 18S rRNA V4 region of target protozoa (Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii) or oyster (Crassostrea virginica), expected amplicon size and GC content for each product.
| Primer IDa | Sequence (5′-3′) |
|---|---|
| General 18S V4 Forward | GCC GCG GTA ATT CCA GCT C |
| General 18S V4 Reverse 1 | ATY YTT GGC AAA TGC TTT CGC |
| Giardia 18S V4 Reverse 2 | ATA CGG TGG TGT CTG ATC GC |
| Target | Gene-specific PCR Amplicon Size (bp) | Gene-specific PCR Amplicon Size with overhangs (bp)a | Dual-indexing PCR Amplicon Size (bp)a | Percent GC |
|---|---|---|---|---|
| T. gondii | 385 | 452 | 521 | 42.0% |
| C. parvum | 366 | 433 | 502 | 28.0% |
| G. enterica | 315 | 382 | 451 | 71.4% |
| C. virginica | 381 | 448 | 517 | 49.6% |
a
Incorporation of overhangs in the gene-specific primer adds 67 bp to the overall fragment length, and incorporation of sequences in dual-indexing PCR adds another 69 bp. However, only the non-overhang, gene-specific amplicon and relevant indices are sequenced.