Last May (the 15th, to be precise), we sent three eppendorf tubes containing Illumina Tru-Seq and NEB-Next libraries constructed from Inga DNAs, most of which had been extracted from herbarium specimens, to the Edinburgh Genomics sequencing facility at the University of Edinburgh. A series of Botanics Stories blogs (listed below) described the rationale for the project, and the methodology that we followed in order to fill these tubes with libraries. Then followed a bit of a hiatus, so it seems that a quick update is in order.
In short: We got the data back on the 1st of June 2015, analysed it – it worked – and wrote a brief paper that was submitted to the journal Taxon on the 4th of February 2016, and accepted on the 19th of May. This was followed by a pleasant celebratory lunch in one of the local bars, the Orchard, on Wednesday the 25th… but back to the data:
Data from Hart et al. (Taxon, in press 2016): The results of the MiSeq runs, by library, are given in the following table. The final row is the silica-dried material from Dexter 401 (E) sequenced by Nicholls & al. (2015), for comparison with libraries from H2004. The library names start with H or S, depending on whether the DNA was extracted from herbarium specimens or silica-preserved tissue samples, followed by the collection year for each accession; the second part of each name reflects the library preparation kit (Tru-Seq or NEB-Next) and whether or not the DNA was repaired (+ or -), with a number that links back to previous blogs on DNA extraction, fragmentation, size selection and library preparation methods.
| Library | No. of trimmed reads | % reads aligned to baits | % reads aligned to Inga plastid | Average quality score of variant positions (AQV) | Number of variant bases | Loci recovered (max 276) | Conservatively called sequence (CCS), bp |
| H1835_NEB13+ | 1013414 | 87.4% | 4.3% | 139.18 | 7186 | 249 | 317244 |
| H1841_NEB07+ | 214315 | 53.9% | 0.7% | 101.80 | 883 | 137 | 46045 |
| H1841_NEB08+ | 365550 | 73.2% | 0.8% | 73.44 | 2773 | 226 | 120148 |
| H1932_NEB11+a | 1226043 | 89.0% | 4.7% | 157.83 | 6377 | 248 | 322337 |
| H1932_NEB11+b | 862599 | 89.1% | 4.1% | 141.89 | 6253 | 246 | 310470 |
| H1932_NEB11+bv2 | 1152606 | 90.0% | 2.3% | 133.15 | 5930 | 248 | 301994 |
| H1932_NEB12+ | 1919229 | 87.4% | 6.4% | 173.56 | 6463 | 249 | 331326 |
| H1948_NEB05+ | 583010 | 87.4% | 1.6% | 94.94 | 5028 | 239 | 241758 |
| H1948_NEB06+ | 704977 | 87.1% | 3.7% | 136.32 | 6132 | 247 | 298809 |
| H2004_NEB09+ | 1787314 | 74.3% | 9.2% | 168.53 | 7018 | 248 | 328618 |
| H2004_NEB10+ | 1595602 | 80.3% | 10.4% | 174.46 | 7135 | 250 | 334242 |
| H2009_NEB01- | 1711918 | 75.0% | 8.6% | 169.24 | 6482 | 248 | 326187 |
| H2009_NEB01+ | 1658799 | 76.6% | 8.2% | 169.21 | 6484 | 250 | 324340 |
| H2009_NEB02- | 1355984 | 75.2% | 8.3% | 163.79 | 6525 | 247 | 322957 |
| H2009_NEB02+ | 1668026 | 76.2% | 8.5% | 171.90 | 6516 | 250 | 326466 |
| H2009_NEB03- | 1513515 | 73.8% | 8.3% | 162.85 | 6463 | 246 | 319683 |
| H2009_NEB03+ | 1504758 | 74.0% | 8.4% | 161.80 | 6419 | 245 | 320273 |
| H1835_Tru13+ | 659161 | 84.2% | 5.2% | 132.97 | 7045 | 247 | 310949 |
| H1932_Tru11+a | 1584437 | 87.7% | 3.8% | 155.89 | 6246 | 248 | 322199 |
| H1932_Tru11+b | 1015706 | 87.5% | 3.8% | 144.88 | 6194 | 248 | 314862 |
| H1932_Tru11+b2 | 1416246 | 87.0% | 4.6% | 159.42 | 6448 | 249 | 324910 |
| H1932_Tru12+ | 1774508 | 84.4% | 6.3% | 169.72 | 6503 | 248 | 330462 |
| H1948_Tru05+ | 1042441 | 83.9% | 2.6% | 136.01 | 5941 | 248 | 296844 |
| H1948_Tru06+ | 892927 | 84.6% | 3.9% | 145.22 | 6211 | 247 | 308853 |
| H2004_Tru09+ | 1958838 | 77.9% | 9.2% | 173.90 | 7041 | 249 | 333904 |
| H2004_Tru10+ | 1576572 | 77.4% | 9.5% | 170.05 | 7066 | 248 | 330278 |
| H2009_Tru01- | 1338317 | 77.6% | 9.1% | 167.51 | 6601 | 249 | 324201 |
| H2009_Tru01+ | 1536759 | 77.2% | 8.4% | 167.03 | 6594 | 248 | 325184 |
| H2009_Tru02- | 1476338 | 76.6% | 8.4% | 166.63 | 6569 | 249 | 323881 |
| H2009_Tru02+ | 1226123 | 75.6% | 8.5% | 161.46 | 6572 | 249 | 319045 |
| H2009_Tru03- | 1630041 | 75.4% | 8.6% | 168.09 | 6509 | 250 | 324451 |
| H2009_Tru03+ | 1753019 | 75.0% | 8.4% | 167.90 | 6512 | 249 | 323951 |
| S2004_TruKD401 | 689439 | 74.4% | 9.2% | 156.29 | 5809 | 245 | 330396 |
References:
Hart, M.L., L.L. Forrest, J.A. Nicholls & C.A. Kidner. In press. Retrival of hundreds of nuclear loci from herbarium specimens. Taxon.
James A. Nicholls, R. Toby Pennington, Erik J.M. Koenen, Colin E. Hughes, Jack Hearn, Lynsey Bunnefeld, Kyle G. Dexter, Graham N. Stone & Catherine A. Kidner. 2015. Using targeted enrichment of nuclear genes to increase phylogenetic resolution in the neotropical rain forest genus Inga (Leguminosae: Mimosoideae). Frontiers in Plant Science 6: 710. doi: 10.3389/fpls.2015.00710
Capturing Genes from Herbaria. I. What it’s all about. http://stories.rbge.org.uk/archives/16411
Capturing Genes from Herbaria. II. Inga. http://stories.rbge.org.uk/archives/16427
Capturing Genes from Herbaria. III. The samples. http://stories.rbge.org.uk/archives/16441
Capturing Genes from Herbaria. IV. DNA. http://stories.rbge.org.uk/archives/16470
Capturing Genes from Herbaria. V. Fragmenting the DNA. http://stories.rbge.org.uk/archives/16525
Capturing Genes from Herbaria. VI. Size Selection. http://stories.rbge.org.uk/archives/16645
Capturing Genes from Herbaria. VII. Comparisons. http://stories.rbge.org.uk/archives/16737
Capturing Genes from Herbaria. VIII. Amplification. http://stories.rbge.org.uk/archives/16788
Capturing Genes from Herbaria. IX. Hybrid capture. http://stories.rbge.org.uk/archives/17298
Capturing Genes from Herbaria. X. An update. http://stories.rbge.org.uk/archives/20751
Capturing Genes from Herbaria. XI. Some metagenomics of a herbarium specimen. http://stories.rbge.org.uk/archives/20817