Both the Tru-Seq and NEB libraries were amplified pre-hybrid capture – another step at which modifications were made, according to how much DNA there was in each library. Bioanalyser traces for the pre-amplification Tru-Seq libraries are shown below:

Bioanalyser traces for size selected Inga umbellifera libraries: unrepaired DNA: 2009 herbarium; 2009 silica; 2009 silica; repaired DNA: 2009 herbarium, 2009 silica, 2009 herbarium, 1948; 1948; 2004 herbarium; 2004 herbarium; 1932 flower; 1932 flower; 1932 leaf; 1985; 1932 flower size select 2

Bioanalyser traces for size selected Inga umbellifera Tru-Seq libraries: unrepaired DNA: 2009 herbarium; 2009 silica; 2009 silica; repaired DNA: 2009 herbarium, 2009 silica, 2009 herbarium, 1948; 1948; 2004 herbarium; 2004 herbarium; 1932 flower; 1932 flower; 1932 leaf; 1985; 1932 flower size select 2

The number of PCR amplification cycles used for both Tru-Seq and NEB for each library are given in the last two columns of the following table:

Year Sample
 DNA repair Sonic cycles Post-sonic cleanup Size Select TruSeq ng NEB ng Library No. Tru-Seq amp cycles NEB amp cycles
2009 H-L – 8 yes normal 101 100 1- 8 7
2009 H-L + 8 yes normal 101 101.5 1+ 8 7
2009 H-L – 8 yes normal 101 100 2- 8 7
2009 H-L + 8 yes normal 70.6 28.2 2+ 9 10
2009 S-L – 8 yes normal 101 100 3- 8 7
2009 S-L + 8 yes normal 101 94 3+ 8 7
2004 H-L + 3 yes normal 101 100 9+ 8 7
2004 H-L + 3 yes normal 70.6 39.1 10+ 9 10
1948 H-L + — — modified 70 22.9 5+ 8 10
1948 H-L + — — modified 80 58.8 6+ 8 7
1932 H-F + — — modified 80 80 11+a 8 8
1932 H-F + — — modified 80 80 11+b 8 8
1932 H-F + — — modified 40 — 11+b2 8 —
1932 H-F + — — — — 40 11+bv2 — 10
1932 H-L + 3 yes normal 101 100 12+ 8 7
1840 H-L + — — — — 5 8+ — 12
1840 H-L + — — — — very low 7+ — 12
1835 H-L + 1 no modified 40.4 16 13+ 10 10

 

Bioanalyser traces for the post-amplification Tru-Seq libraries are shown below:

Bioanalyser traces for amplified Inga umbellifera libraries: top row – unrepaired DNA: 2009 herbarium; 2009 silica; 2009 silica; repaired DNA: second row – 2009 herbarium, 2009 silica, 2009 herbarium; third row – 1948; 1948; 2004 herbarium; fourth row – 2004 herbarium; 1932 flower; 1932 flower; bottom row – 1932 leaf; 1835; 1932 flower size select 2

Bioanalyser traces for amplified Inga umbellifera Tru-Seq libraries: top row – unrepaired DNA: 2009 herbarium; 2009 silica; 2009 silica; repaired DNA: second row – 2009 herbarium, 2009 silica, 2009 herbarium; third row – 1948; 1948; 2004 herbarium; fourth row – 2004 herbarium; 1932 flower; 1932 flower; bottom row – 1932 leaf; 1835; 1932 flower size select 2

The peaks of the amplified TruSeq libraries are far higher, although the first six libraries also show a small peak at around 2x the main peak size. It is possible that this is a product of slight over-amplification of the libraries. Initially we wondered if it could have been due to a small carry-over of beads, but replacing the tubes on the magnet and repipetting the sample off did not remove or reduce this peak. There is also a small adaptor peak visible in some of the traces at c. 120 bp.

For the NEB libraries, we did not generate BioAnalyser traces pre-amplification. The post-amplification NEB Bioanalyser traces are shown below, for comparison with the Tru-Seq traces above:

Bioanalyser traces for size selected Inga umbellifera NEB libraries: top row – unrepaired DNA: 2009 herbarium; 2009 silica; 2009 silica; repaired DNA: second row – 2009 herbarium, 2009 silica, 2009 herbarium; third row – 1948; 1948; 2004 herbarium; fourth row – 2004 herbarium; 1932 flower; 1932 flower; bottom row – 1932 leaf; 1835; 1932 flower size select 2

Bioanalyser traces for size selected Inga umbellifera NEB libraries: top row – unrepaired DNA: 2009 herbarium; 2009 silica; 2009 silica; repaired DNA: second row – 2009 herbarium, 2009 silica, 2009 herbarium; third row – 1948; 1948; 1840; fourth row – 1840; 2004; 2004; fifth row – 1932 flower; 1932 flower; 1932 leaf; bottom row – 1835; 1932 flower size select 2

A small adaptor peak is visible at c. 60 bp. One 1840 library (final sample on row three) was generated from far less than the minimum recommendation of 5 ng of repaired DNA for the NEB protocol we were following; the other 1840 library (first sample on row four) was generated from 5 ng of repaired DNA, and clearly has a far better DNA fragment size distribution.

 

We then sorted all 32 Tru-Seq and NEB post-amplification libraries into three pools of libraries with compatible adaptor sequences, taking into account average library fragment size, for three separate hybrid capture reactions.

Pool 1: 439-472 bp: no DNA repair: Tru-Seq and NEB, 2009 herbarium and 2009 silica; DNA repair: Tru-Seq and NEB, 2009 herbarium and 2009 silica.

Pool 2: 450-520 bp: no DNA repair: Tru-Seq and NEB, 2009 herbarium; DNA repair: Tru-Seq and NEB, 2004 herbarium, 1932 leaf; Tru-Seq, 1932 flower (size select 2).

Pool 3: 350-400 bp: DNA repair: TruSeq and NEB, 1948, 1932 flower, 1835; NEB, 1840, 1932 flower (no size select).

 

 

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