Table 2.
454 library quantification and sequencing results obtained from fifteen samples
| Sample | Library construction | 454 sequencing | ||||||
|---|---|---|---|---|---|---|---|---|
| ID | Initial material (ng) | Mean fragment size (bp) | Concentration (molec./μl) | Recovery (%) | Copies per bead in emPCR | Enriched beads | Mixed sequences (%) | Filter passed sequences |
| Neandertal 1 | n/a | 200 | 2.00 × 106 | n/a | 2.17 | 144 275 | 8.7 | 15 541 |
| Neandertal 2 | n/a | 200 | 1.75 × 107 | n/a | 2.25 | 98 310 | 9.5 | 16 972 |
| Neandertal 3 | n/a | 200 | 1.88 × 107 | n/a | 2.08 | 109 330 | 8.2 | 16 447 |
| Neandertal 4 | n/a | 200 | 1.82 × 107 | n/a | 2.17 | 78 010 | 9.2 | 16 773 |
| Neandertal 5 | n/a | 200 | 5.48 × 106 | n/a | 2.19 | 101 790 | 9.4 | 17 323 |
| Neandertal 6 | n/a | 200 | 7.40 × 107 | n/a | 2.16 | 58 580 | 11.3 | 13 590 |
| Neandertal 7 | n/a | 200 | 9.74 × 107 | n/a | 2.14 | 55 680 | 10.8 | 19 639 |
| Neandertal 8 | n/a | 200 | 4.88 × 107 | n/a | 2.20 | 62 640 | 9.5 | 14 062 |
| Neandertal 9 | n/a | 200 | 3.99 × 107 | n/a | 2.15 | 62 640 | 11.5 | 15 582 |
| Neandertal 10 | n/a | 200 | 1.45 × 108 | n/a | 1.59 | 26 100 | 7.8 | 17 030 |
| Neandertal 11 | n/a | 200 | 7.85 × 107 | n/a | 2.33 | 54 230 | 8.9 | 14 592 |
| Neandertal 12 | n/a | 200 | 4.31 × 107 | n/a | 2.25 | 60 320 | 9.2 | 16 544 |
| SAGE ditags | 32 | 250 | 9.83 × 107 | 0.5 | 1.64 | 76 270 | 3.4 | 21 429 |
| Amplicons | 85 | 320 | 1.72 × 108 | 0.4 | 1.35 | 42 000 | 11.2 | 20 716 |
| Bonobo | 520 | 450 | 1.50 × 109 | 1.7 | 2.33 | 87 800 | 20.3 | 12 232 |
No attempts were made to quantify the Neandertal DNA extracts prior to library preparation in order to preserve precious sample. Library concentrations were inferred by qPCR taking into account the mean fragment size of each library. The percentages of molecules recovered after library preparation are denoted. A narrow range of copy per bead ratios was used in emPCR. The success of emPCR is determined both by the number of enriched beads obtained (50 000 beads required) and the ratio of mixed sequences. Due to the highly fragmented nature of ancient DNA, the run-processing parameters were changed for the Neandertal samples in order to accept shorter reads (see Supplementary Data). Subsequently, emPCR artefact sequences were removed as described in Ref. (11). The other samples were processed with the default run-processing parameters. All plate regions yielded more than 12 000 sequences after filtering.