Real-time quantitative PCR (qPCR) amplification
The primer design was performed using the in-house primerXL pipeline, which uses
empirically validated design rules and performs a thorough downstream primer evaluation.
The following parameters were used: minimal overlap of 20 bp between amplicons covering
the same exon, 40 bp up- and downstream included intronic sequence, and no SNPs (dbSNP
version 130) or secondary structures allowed in the primer annealing sites. In addition, nonspecific primers were excluded. In case no primers could be found according to the
parameters set, the maximal amplicon length was increased. Primer sequences are available
on request.
The total qPCR reaction volume of 5 l contained 2.5 l SsoFast EvaGreen Supermix (2x)
(Bio-Rad), 0.5 l template gDNA (7 ng/l) and 2 l F and R primermix (0.625 M working
solution). Cycling conditions comprised 5 min at 98°C, 45 cycles of 15 sec at 98°C, 30 sec at
60°C and 1 min 30 sec at 72°C, followed by a dissociation curve analysis from 60°C to 95°C
(LightCycler 480, Roche). The Cq value of each qPCR represents the fractional PCR cycle at
which the fluorescence of the sample rises above the background fluorescence.
Ligation and shearing
First, 240 l of each PCR pool was purified and eluted to a final volume of 34 l using three
spin-columns of the QIAquick PCR Purification kit (Qiagen). Subsequently, a DNA endrepair reaction was performed on 25 l of the purified PCR product in a standard 50 l
reaction (End-it DNA End-Repair kit, EpiCentre Biotechnologies). The reaction mix was
incubated for 45 min at 21°C followed by 10 min at 70°C. The resulting product was purified
and eluted to 30 l (QIAquick PCR Purification kit, Qiagen). Finally, two aliquots of 10 l of
the purified end-repaired DNA were ligated using two standard 15 l reactions for blunt-
ended DNA (Fast-Link DNA Ligation kit, EpiCentre Biotechnologies). Ligation was
performed overnight (13h) at 16°C and inactivated for 15 min at 70°C.
Next, 25 l of the total ligated product was diluted to 120 l with TE buffer (Invitrogen) in
Covaris microTubes (6x16mm) and sheared to a target length of 300 bp using the following
conditions: duty cycle of 10%; intensity of 4; 200 cycles per burst; 120 sec (Covaris S2). The
sheared DNA was then purified (QIAquick PCR purification kit, Qiagen).
Quality control during the protocol consisted of DNA concentration measurement on a ND1000 NanoDrop (Thermo Fisher Scientific) following each purification step prior to ligation.
In addition, the size of ligated and sheared products was evaluated on a 2100 Bioanalyzer
with the DNA7500 and DNA1000 kits (Agilent), respectively, and concentrations were
determined using the Quant-iT PicoGreen dsDNA assay (Invitrogen) on the FLUOstar
OPTIMA (BMG Labtech).
Consolidation and error correction methods of condensation
The NextGene condensation tool corrects low frequency errors by generating a consensus
sequence from clustered reads. In specific, Illumina reads are clustered in groups based on the
presence of a common anchor sequence of 12 bp. In a next step, subgroups are generated
based on the anchor’s flanking shoulder sequences. Subsequently, a consensus sequence is
generated from each subgroup, taking into account the position of each base in the reads (with
base calls on the 5’end having a higher weight than on the 3’end).
In the consolidation method of condensation, overlapping sequences are merged and the
consensus sequence is used in place of all the original reads of the subgroup. In the error
correction method of condensation, the original reads are maintained, with low frequency
errors corrected.