Supplementary Figure 1 Concept of barcoding to suppress error in sequencing. Each template DNA molecule is barcoded with a random and unique sequence (marked as red, turquoise and green). All PCR generated molecules that are generated from the same original molecule receive the same barcode. If a polymerase induced error occurs (blue cross), only a fraction of all DNA molecules with the same barcode will amplify that specific error. Conversely, a template DNA molecule with mutation will generate PCR amplicons with that particular barcode and can therefore be called a true mutant.
Supplementary Figure 2 Organization of SiMSen-Seq analysis data produced by Debarcer. The Debarcer output directory contains files in the main output directory (denoted Top Level ) and two subdirectories found in this location (Tables directory and Figures directory). Files described by each unshaded box in the figure can be found in their respective directories.
Supplementary Figure 3 Fragment analysis of single-plex library at low DNA input. Unpurified libraries were generated for a target amplicon in the CD1C gene using 10, 5 and 3 ng of high quality human genomic DNA (left side) or fragmented plasma DNA (right side) along with a no DNA control (bottom). Expected library products are indicated by the horizontal bar while all other products (<200bp) are nonspecific. Clear library PCR products can be seen in all plots except the no DNA control.
Supplementary Figure 4 Fragment analysis of triplex library at low DNA input. Unpurified libraries were generated for three target amplicons in the CD1C, ERBB4, and COL5A1 genes using 10, 5 and 3 ng of high quality human genomic DNA (left side) or fragmented plasma DNA (right side) along with a no DNA control (bottom). Expected library products are indicated by the horizontal bar while all other products (<200bp) are non-specific. Clear library PCR products can be seen in all plots except the no DNA control.
Supplementary Figure 5 Schematic representation of the first three rounds of PCR used to incorporate barcodes. Figure shows PCR products that are formed in cycles 1, 2 and 3 of the barcoding PCR step. Note that only DNA strands with both forward and reverse adapter sequences (or their complement) can act as templates in the second round of PCR. These products are indicated with thicker lines.
Supplementary Table S1. Adapter primer sequences. Name Primer sequence (5 3 ) Universal forward adapter primer AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT Reverse index 1 adapter primer CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 2 adapter primer CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 3 adapter primer CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 4 adapter primer CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 5 adapter primer CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 6 adapter primer CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 7 adapter primer CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 8 adapter primer CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 9 adapter primer CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 10 adapter primer CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 11 adapter primer CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 12 adapter primer CAAGCAGAAGACGGCATACGAGATTACAAGGTGACT GGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 13 adapter primer CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 14 adapter primer CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 15 adapter primer CAAGCAGAAGACGGCATACGAGATTGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 16 adapter primer CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 17 adapter primer CAAGCAGAAGACGGCATACGAGATCTCTACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 18 adapter primer CAAGCAGAAGACGGCATACGAGATGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 19 adapter primer CAAGCAGAAGACGGCATACGAGATTTTCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 20 adapter primer CAAGCAGAAGACGGCATACGAGATGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 21 adapter primer CAAGCAGAAGACGGCATACGAGATCGAAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 22 adapter primer CAAGCAGAAGACGGCATACGAGATCGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 23 adapter primer CAAGCAGAAGACGGCATACGAGATCCACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 24 adapter primer CAAGCAGAAGACGGCATACGAGATGCTACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 25 adapter primer CAAGCAGAAGACGGCATACGAGATATCAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 26 adapter primer CAAGCAGAAGACGGCATACGAGATGCTCATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 27 adapter primer CAAGCAGAAGACGGCATACGAGATAGGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 28 adapter primer CAAGCAGAAGACGGCATACGAGATCTTTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 29 adapter primer CAAGCAGAAGACGGCATACGAGATTAGTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 30 adapter primer CAAGCAGAAGACGGCATACGAGATCCGGTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 31 adapter primer CAAGCAGAAGACGGCATACGAGATATCGTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 32 adapter primer CAAGCAGAAGACGGCATACGAGATTGAGTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 33 adapter primer CAAGCAGAAGACGGCATACGAGATCGCCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 34 adapter primer CAAGCAGAAGACGGCATACGAGATGCCATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 35 adapter primer CAAGCAGAAGACGGCATACGAGATAAAATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 36 adapter primer CAAGCAGAAGACGGCATACGAGATTGTTGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 37 adapter primer CAAGCAGAAGACGGCATACGAGATATTCCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 38 adapter primer CAAGCAGAAGACGGCATACGAGATAGCTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 39 adapter primer CAAGCAGAAGACGGCATACGAGATGTATAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 40 adapter primer CAAGCAGAAGACGGCATACGAGATTCTGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 41 adapter primer CAAGCAGAAGACGGCATACGAGATGTCGTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 42 adapter primer CAAGCAGAAGACGGCATACGAGATCGATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 43 adapter primer CAAGCAGAAGACGGCATACGAGATGCTGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 44 adapter primer CAAGCAGAAGACGGCATACGAGATATTATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 45 adapter primer CAAGCAGAAGACGGCATACGAGATGAATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 46 adapter primer CAAGCAGAAGACGGCATACGAGATTCGGGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 47 adapter primer CAAGCAGAAGACGGCATACGAGATCTTCGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Reverse index 48 adapter primer CAAGCAGAAGACGGCATACGAGATTGCCGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Sample index barcode indexes are shown in red