Fig. 1

JTax workflow and reference rearrangement. a Given a pair of forward (blue) and reverse (red) primers, DNA samples are amplified and the products are subjected to Illumina PE sequencing (left panel). Color gradient indicates 5′ (dark; high quality) to 3′ (light; low quality) end of a read. Right and left arrows indicate reads on the plus and minus strand respectively. PE reads are then joined either directly or in an inside-out manner. The right panel shows rearrangement of a reference sequence in the two joining methods (see main text for details). b Identification of primer sites on a reference. Based on alignment between the main reference (blue) and a reference sequence (green), base positions on the reference are converted into coordinates on the main reference (i.e., 12, 13, 14, 14, 15, …, 34, 35, 37, 38, …). Primer sites on the main reference (in rectangles) are then obtained using USEARCH (command: search_oligo, option: –maxdiffs 3). At the forward primer site, JTax scans from the reference start the first coordinate (i.e., 12, in red) greater than the primer start (i.e., 10, in blue). To reach the primer start, it attempts to back trace two bases on the reference. However, no bases are found on the left of the corresponding position, so two Ns are padded. For the reverse primer site, the first coordinate smaller than the primer end (i.e., 36, in blue) is 35 when scanning from the reference end. JTax then extends one base from the corresponding position (i.e., 57, in red) on the reference to 58 (in green). The resulting amplicon is “NNAGTCTTGA … CGAGGTAA”