As previously mentioned, we tested two different kits in our NBAF project. The first is the Illumina Tru-Seq Nano library preparation kit (FC-121-4001), which recommends a starting DNA quantity of 100 ng. We also used NEB‘s NEBNext Ultra library preparation kit for Illumina (E7370S), which has been optimized for as little as 5 ng of starting DNA. The DNA for each kit was aliquoted into separate tubes, and each sonicated separately just prior to library preparation, using the same sonication profile for both aliquots. The amounts of DNA given below are the quantity that went into the initial library preparation, after DNA repair; this starting point varied according to whether libraries were sonicated or not.

For one collection (1940 herbarium) there was extremely little DNA – for this, only the NEB library was made.

Year Material type Tissue Extract DNA repair Sonicat. cycles Post sonicat. cleanup Size select TruSeq ng NEB ng Library no.
2009 Herbarium Leaf DNeasy no 8 yes normal 101 100 1-
2009 Herbarium Leaf DNeasy yes 8 yes normal 101 101.5 1+
2009 Herbarium Leaf QiaQuick no 8 yes normal 101 100 2-
2009 Herbarium Leaf QiaQuick yes 8 yes normal 70.6 28.2 2+
2009 Silica Leaf DNeasy no 8 yes normal 101 100 3-
2009 Silica Leaf DNeasy yes 8 yes normal 101 94 3+
2004 Herbarium Leaf DNeasy yes 3 yes normal 101 100 9+
2004 Herbarium Leaf both yes 3 yes normal 70.6 39.1 10+
1948 Herbarium Leaf DNeasy yes none — modified 70 22.9 5+
1948 Herbarium Leaf DNeasy yes none — modified 80 58.8 6+
1932 Herbarium Flower DNeasy yes none — modified 80 80 11+a
1932 Herbarium Flower DNeasy yes none — modified 80 80 11+b
1932 Herbarium Flower DNeasy yes none — modified 40 — 11+b2
1932 Herbarium Flower DNeasy yes none — none — 40 11+bv2
1932 Herbarium Leaf DNeasy yes 3 yes normal 101 100 12+
1840 Herbarium Leaf DNeasy yes none — none — 5 8+
1840 Herbarium Leaf DNeasy yes none — none — very low 7+
1835 Herbarium Leaf DNeasy yes 1 no modified 40.4 16 13+

For one of the 1840 library preparations, the quantity of DNA in the aliquot was too low to measure using a Qubit dsDNA high sensitivity kit.

 

 

 

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