King's Next-Generation Sequencing Meeting
Michelle Lupton
Additional references

Linnarsson S. Recent advances in DNA sequencing methods - general
principles of sample preparation. Exp Cell Res. 2010 May 1;316(8):1339-43.
Epub 2010 Mar 6. Review. PubMed PMID: 20211618.

Buehler B, Hogrefe HH, Scott G, Ravi H, Pabón-Peña C, O'Brien S, Formosa R,
Happe S. Rapid quantification of DNA libraries for next-generation
sequencing. Methods. 2010 Apr;50(4):S15-8. Review. PubMed PMID:
20215015.-the use of real time PCR
Stages in the library preparation
Steps accompanied by numbers are those for which we suggest alternatives to
the standard Illumina protocols. Numbers correspond to those given in
Supplementary Protocols
Fragmentation

Nebulization
Uneconomical distribution of fragment size. Approximately half of the DNA vaporises

Adaptive Focused acoustics – Covaris
Acoustic energy is controllably focused into the aqueous DNA sample by a dish-shaped transducer,
resulting in cavitatin events within the sample.
17% of the sample is in the 200bp size range, and little DNA loss
Fragmentation
Enzymatic digestion (Linnarsson 2010)



Two recent commercial enzymatic fragmentation kits were introduced.
Fragmentase (New England Biolabs) - based on V.vulnificus nuclease that generates
random nicks, and modified T7 endonuclease that recognises the nicks and cleaves the
opposite strands.
Nextera (Epicentre) - based on random transposon insertion. Also introduces adapter
sequences simultaneously with fragmentation.
A-tailing, ligation and size
selection

Artefacts from standard library prep;
1.
3.
Bias in base composition
High frequency of chimeric sequences
Imperfect insert size distribution

Overcome by;
1.
Pair-end oligos
Gel extraction-melt slice at room temp-reduces GC bias
Improved efficiency of the end repair and A-tailing
Double size selection
Paired end size selection-only excise a 2mm size gel slice
2.
2.
3.
4.
5.
Figure 3
A-tailing, ligation and size
selection
GC plots before (a) and after
(b) optimisation of gel
extraction. The figures show
the total area in which reads
with a particular GC content
are distributed, with the
mean and standard
deviation. The greater width
of shaded area in plot a)
indicates a wider dispersion
of coverage for all values of
GC content for which
sequences were obtained.
Agilent traces Bioanalyzer
2100 traces for two
suboptimal libraries c) 60bp
insert library, with optimised
PCR, d) the same 60bp
library with excess DNA in
PCR e) 200bp insert library,
showing shoulder of small
fragments.
Insert size distribution from
sequenced human DNA
using f) the standard and g)
modified paired end library
prep protocols
PCR

Template quality -use optimized quantities of DNA template.

Use of high fidelity polymerases in an optimised reaction.

Use of solid phase reversible immobilization SPRI technology (SPRI) removes a higher
proportion of primers and adapter dimers than spin columns.

Reduce the number of PCR cycles: 3ng DNA and 14 cycles of PCR amplification for
single end libraries, 25ng DNA and 12 cycles for high complexity libraries, and 10ng
DNA and 18 cycles for lower complexity samples. These quantities give the optimal
compromise between clean libraries and a low frequency of duplicate sequences.

Possible to eliminate the PCR step by ligating on appropriate adaptors after Atailing.

Direct sequencing of short amplicons.
Figure 4
PCR
a) A ~200bp fragment
library was prepared, and
10ng was amplified for 18
cycles using standard
Illumina conditions, and
with more optimal PCR
conditions.
b) After PCR we divided
the library into two: half
was purified following the
standard Illumina protocol,
through a Qiaquick PCR
cleanup column, whereas
the other was purified
using SPRI technology.
Each was then run on an
agarose gel alongside a
100bp ladder to view the
DNA species that
remained.
PCR

LIBRARY
KPOA0006
KPOC0002
KPOA0005
KPOC0001
NA18507
KPOC0005
KPOA0010
KPOA0008
KPOC0004
KPOC0003
KPOA0017
KPOA0014
PCR duplication example;
UNPAIRED
READS
EXAMINED
62519
57432
45961
51485
21791
54337
25997
48848
38812
201355
74130
52506
READ
PAIRS
EXAMINED
5464702
3448312
3143590
2884859
2187848
3648741
2449286
3474580
2350763
4528357
3097782
3493530
UNMAPPED
READS
986509
624078
562785
547339
369343
681663
410855
628848
396988
1027539
600782
618672
UNPAIRED
READ
DUPLICATES
47544
33414
21530
32763
15030
47073
17482
33125
24710
137213
48333
37310
READ
PAIR
DUPLICATES
2657808
542256
351362
596494
707778
2802655
711382
1017855
528626
2028173
1114520
1206707
READ
PAIR
OPTICAL
DUPLICATES
15731
16503
9954
10627
7613
14976
8187
10306
8809
23747
15594
11793
PERCENT
DUPLICATION
0.487918
0.160759
0.114359
0.210567
0.325319
0.768841
0.292461
0.295632
0.228246
0.452963
0.363235
0.348136
ESTIMATED
LIBRARY
SIZE
3598454
10024617
13316441
6055683
2620318
858549
3376826
4734070
4462098
3410538
3218521
3829677
Pre
hybridisation
PCR
cycles
5
5
5
5
5
6
5
5
5
5
5
5
Post
hybridisation
PCR
cycles
16
12
16
12
12
12
12
12
12
12
12
12
Quantification

Optimal concentration range of DNA that will
yield clusters in the optimal density range.

Spectrophotometry is not accurate.
From
[bp]
200
Corr.
To [bp] Area
1,000
232.6
% of
Total
79
Average
Size [bp]
375
Size
distribution in
CV [%]
Conc. [pg/µl]
24.1
Molarity
[pmol/l]
165.15
714.3
 Quantitative PCR. Quantify unknown libraries against standard libraries that have been
sequenced previously for which cluster number is known.
Electrophoresis with Agilent bioanalyser
-Gives a check of size distribution.
-Can be inaccurate for a small proportion of libraries, may be due to single stranded DNA not
easily quantified when mixed with double stranded
-Can use the bioanalyser to check size distribution and Fluorometery to determine the
concentration more accurately (e.g. Qubit dsDNA BR Assay)

Quantification
a) Cluster throughput as a
function of total clusters for
200 and 500bp inserts. The
500bp inserts underwent
fewer cycles of cluster
amplification (28, compared
to 35 for the 200bp
libraries), resulting in
smaller clusters, and so a
cluster density of 40-44k /
tile (GA1) will produce the
maximum yield from either
insert size.
b) Standardisation of cluster
density with qPCR
quantification. Runs were
grouped into 25-run bins
and a boxplot plotted. After
some initial problems with
degradation of standards,
cluster number has levelled
out at ~35-40k / tile.
Denaturation
For low concentrations of Double stranded DNA denaturation by heating can
damage DNA and introduce G+C bias.

Use Modified hybrization buffers; prefer use of 0.1NaOH to heating.

Subnanomolar libraries require an alternative buffer.
Addition of Tris to illumina buffer prevents rise in pH.
Diluting supplied 2M NaOH and using a greater volume reduces fluctuation
caused by pipetting error.
1.
2.
Denaturation
a) pH titration of hybridisation buffers.
The concentration of NaOH in DNA
templates is 0.1M NaOH. Adding more
than 8μl of this denatured template to
the 1ml of Hybridisation Buffer prior to
loading DNA onto the flowcell,
increases the pH to above 10. This
prevents efficient hybridisation, and
thus the cluster density falls. The
addition of Tris-HCl pH7.3 to the
supplied bottles of Hybridisation Buffer
dramatically increases buffering
capacity, making template hybridisation
more robust.
b) the addition of 5mM Tris-HCl pH 7.3 to
Illumina Hybridisation Buffer allows a
greater volume of denatured template
to be added before high pH prevents
effective annealing of templates to the
oligos on the flowcell surface. This
increases the robustness of cluster
generation, by counteracting pipetting
errors in the denaturation step.
Amplification Quality control

After cluster amplification double stranded DNA on the flow cell can be
stained using an intercalating dye to be detected by a fluorescence
microscope.

Use on flow cells before linearization and blocking to confirm that the
cluster density is appropriate.
Additions to the method





Careful DNA quantification before fragmentation and checking for degraded DNA.
Use of low absorbing plastic ware (Linnarsson 2010), e.g Beckman Coulter “non stick” or
equivalent. Also advise to add some detergent (e.g. 0.02% Tween-20) to reduce absorption
to tube walls.
The implementation of SPRI XP beads for all purification steps.
The use of the bioanalyser to check concentration and size distribution after fragmentation.
Cheaper alternatives to illumina kits, e.g. NEB kits, making own adapters and primers.
Conclusion

The Genome Analyzer is a powerful sequencing technology, Here the authors
describe a number of modifications that allow for more efficient library
preparation, and which enable a stable workflow in a production environment.

At the Sanger Institute, they have several teams for every stage of
sequencing. All steps in the process are recorded using custom-written labtracking and run-tracking database software.

Combined with improvements to the image analysis software and a faster run
time, they predicted that by Christmas 2008, their output will reach 6-10
terabases of high-quality sequence per year - equivalent to 180 human
genomes at 15-fold coverage, or approximately 200,000 bases per second.

The improved workflow and high yield should maintain the Genome Analyzer
as their next-generation sequencing platform of choice for the immediate
future. But how long this remains true depends upon the performance of
existing rival technologies, and those that are on the horizon. For example
Oxford Nanopore Technologies, and Pacific Biosciences’ Single Molecule
Real Time technology which promise to bring us closer to the eagerly
anticipated $1,000 genome.