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Fig. 4.
NEBNext Ultra DNA reagent calculator.
Fig. 5.
Automated library construction plate layout.
Fig. 6.
E. coli
K12 gDNA control libraries created with the NEBNext
Ultra DNA automated method.
In addition to the user interface, the automated NEBNext
Ultra DNA method provides the user with an HTML-
driven reagent calculator that provides the user with the
final volumes of all of the reagents and master mixes
required on the deck, as well as instructions on how to
generate the various master mixes—based upon the
number of samples to be processed and the amount of
the workflow that the user wishes to pursue. An image
of the reagent calculator is presented in Figure 4.
Results
Sixteen genomic DNA (gDNA) samples from
A. thaliana
and
H. sapiens
, 2
C. elegans
amplicon pools, 2
H. sapiens
ChIP DNA, and 4 bacterial gDNA samples were supplied
by various laboratories at Indiana University, Bloomington,
for Biomek automated library construction at Indiana
University. In addition,
E. coli
K12 gDNA was supplied
by New England Biolabs as a positive control. The 200 ng
aliquots for each of the gDNA samples were sheared
to an average size of 400 bp (data not shown) and arrayed
in the sample plate as shown in Figure 5.
Libraries were constructed using the NEBNext Ultra DNA
automation method utilizing the 400–500 bp insert size
selection option. Eight cycles of PCR enrichment were
used to amplify the libraries using an off-deck thermocycler.
Following analysis on the Agilent 2200 TapeStation, all
of the libraries processed were then sequenced on an
Illumina MiSeq
®
using a 2x300 cycle paired end run.
For the purposes of this document, we concentrated
our analysis on the six
E. coli
K
1
2 control libraries, which were
assayed on the Agilent 2200 TapeStation using D
1
000
ScreenTape (PN# 5067-5582). The electropherograms
for all 6
E. coli
K
1
2 control libraries are presented in Figure 6.
Data analysis was performed at New England Biolabs
using a local instance of Galaxy. For each of the 6
E. coli
K
1
2 control libraries, over
1
.4 million pass filter reads
for each library were generated by the MiSeq run. Pass
filter read counts are presented in Figure 7. Reads were
then trimmed using SeqPrep prior to mapping back to
the
E. coli
K
1
2 MG
1
655 reference genome using Bowtie
(version 2.
1
.
1
0). Less than
1
% of the reads from the
E.
coli
K
1
2 control libraries failed to map back to the
E. coli
K
1
2 MG
1
655 reference genome. We also observed low
percentages of chimeric reads, PCR duplicates, and
mismatched reads. These metrics indicate that the libraries
were high-quality libraries. Data on basic library quality
metrics are presented in Figure 8. Additionally, we
investigated the average size of the library inserts of
the
E. coli
K
1
2 control libraries utilizing the library
mapping data. As shown in Figure 9, the average size
inser ts of the
E. coli
K
1
2 control libraries was
approximately 400 bp.
AAG-350TCH08.14-A