Table 1. Primers, MIDs, sequence formats and consensus reference sequence used in this study.
| Primer | Sequence (5′ - 3′) |
| ColFol-for | TTTCAACAAATCATAARGAYATYGG |
| ColFol-rev | TAAACTTCNGGRTGNCCAAAAAATCA |
| 454-ColFol-for | Adaptor A+MID+TTTCAACAAATCATAARGAYATYGG |
| 454-Col307-rev | Adaptor B+CANCCNGTNCCNGCNCCNCTYTC |
| Raw 454 sequence after demultiplexing | >GGQCYR401C2J7Z length = 354 xy = 1142_0845 region = 1 run = R_2010_05_04_08_38_41_ TTTCAACAAATCATAAGG ATATTGGAACAATATATCTAATACTAGGATCCTGATCAGCTTTTATAGGGACTGCTTTTAGTATCCTGATCCGTATAGAACTAGGCCAACCTGGGACCCTGATTGGAAATGATCAAATCTACAATGTTATGGTGACTGCTCATGCTTTTTGTAATAATTTTCTTTATAGTTATACCAATTATGATTGGAGGGTTTGGGAATTGATTAGTCCCCCTAATAATTGGGGCTCCTGATATAGCCTTCCCACGTATAAATAATATAAGTTTCTGATTACTCCCCCCTTCCCTTACCTTATTAGTCGCGGGAGGTTTAGTAGAAAGAGCGGCAGGAACAGGA |
| Unique sequence output from UniqueSequence.pl | >GGQCYR401C2J7Z_1 ATATTGGAACAATATATCTAATACTAGGATCCTGATCAGCTTTTATAGGGACTGCTTTTAGTATCCTGATCCGTATAGAACTAGGCCAACCTGGGACCCTGATTGGAAATGATCAAATCTACAATGTTATGGTGACTGCTCATGCTTTTTGTAATAATTTTCTTTATAGTTATACCAATTATGATTGGAGGGTTTGGGAATTGATTAGTCCCCCTAATAATTGGGGCTCCTGATATAGCCTTCCCACGTATAAATAATATAAGTTTCTGATTACTCCCCCCTTCCCTTACCTTATTAGTCGCGGGAGGTTTAGTAGAAAGAGCGGCAGGAACAGGA |
| PyroClean output sequence | >Seq1_2343 ATATTGGAACAATATATCTAATACTAGGATCCTGATCAGCTTTTATAGGGACTGCTTTTAGTATCCTGATCCGTATAGAACTAGGCCAACCTGGGACCCTGATTGGAAATGATCAAATCTACAATGTTATGGTGACTGCTCATGCTTTTTGTAATAATTTTCTTTATAGTTATACCAATTATGATTGGAGGGTTTGGGAATTGATTAGTCCCCCTAATAATTGGGGCTCCTGATATAGCCTTCCCACGTATAAATAATATAAGTTTCTGATTACTCCCCCCTTCCCTTACCTTATTAGTCGCGGGAGGTTTAGT |
| Consensus reference sequence | >EMBOSS_001 NHNNNNNTNNNTNNWNHTNKSNNNNNKNNNNNSNHYNNYNGGNDYNDNNYTNARNNYNNNNNTNNSNNNNRANNTNRSNVRNNYNRGNNNNNWNNTNRRNNRNGANCANVYNTANAAYRYNNYDRTNACNKCNCANGCNKTYDYNATRATNTTYTTYRYDGTNAKNCCNNTHWTRVTHGGNGGNHTHGGNAANTKRHTNVTNCCNNTNATRVTNRRNKCNSCNGAYATNKCNTTNCCNCGNHTNANNAAYHTRAGNTTYTGRYTNYTNCCNCCNDSNHTNNNNNTNNTNNBNNNNRGNDSNNYNDBNNANDNNGRNDNNGGNACNGGNTGRNNNNYNTAYCCNCCNNTNKCNDVNNNNNYNDBNCANNNNGGNNBNDSNRTNGANNTNDNNATYTTYWSNYTNCANYYNRCNGGNRYNNSNTMNATYYTNGGNGCNRTNARYTTYANNWSNWCNDBHDDNNAYATNNRNNNNNNNNNNNTNNNNTGRRANNDNNYNHBNYTNYTNNBNTGNDSNRTNHWHNTNACNDCNDYHYTNYTNBYNNYNDSNHTNCCNKTNNTNNNNGGNGCNRTNWCNATRYTNNTNWYNGAYCGNAANNTNAANNCNDSNTTYTTYNNNCCNDSNGGNGGNGNNGANYMNRWHYTNTWNCANCNYHWNNYY |
ColFol-for ColFol-rev are the Sanger primers. 454-ColFol-for and 454-Col307-rev are the primers for mass amplification. Adaptors A and B are used by the ‘454’ sequencer to attach individual DNA molecules to microscopic beads, for subsequent sequencing. MIDs (Multiplex Identifiers) are 7 bp sequences that allow different samples to be sequenced together on a single ‘454’ plate and then separated bioinformatically for downstream analysis. There is no MID with 454-Col307-rev because we only pyrosequenced from the forward direction. Row 5 is an example of a 454 read after demultiplexing with the Roche tools. The forward primer is underlined, and the reverse primer dashed underlined. The MID tag is removed during demultiplexing. Row 6 is an example of a sequence after processing of sequences to produce a file of unique sequences.