Development of a PCR-free protocol for the sequencing library
preparation
Two parameters are critical for adapting the SSV-Seq library preparation
protocol to a PCR-free method: (i) the reduction of the number of steps
to avoid DNA loss during beads-based cleanup and (ii) the use of
appropriate adapters (Figure 1 ). SSV-Seq adapters are suitable
for use in a PCR-free protocol (Kozarewa et al., 2009) because they
contain all elements required for the bridge amplification on Illumina
flow cells, i.e. the sequences complementary to the flow cell
oligonucleotides, the sequence targets of the P5/P7 sequencing primers
and 6 base-indexes. Among PCR-free kits that are commercially available,
6 kits were first selected because of their compatibility with Illumina
technology (Supplementary Table S3) . Three out of 6 kits were
tested, i.e. KAPA HyperPrep (Roche), NxSeq AmpFREE Low DNA (Lucigen) and
NEBNext Ultra II (New England Biolabs) based on the following criteria:
(i) a low amount of fragmented DNA (≤ 200 ng) is required as input, (ii)
the end repair and A-tailing steps are combined into a unique step,
(iii) home-made adapters can be used and (iv) kits are compatible with
Illumina paired-end sequencing. Libraries were prepared from 200 ng of
fragmented PhiX174 DNA following the instructions of each kit, at the
exception of two steps: the SSV-Seq adapters were used for the ligation
instead of the commercial ones, and the post-ligation cleanup and size
selection steps were replaced by a double purification with 1X SPRI
beads (Figure 1, SSV-Seq 2.0 ). Libraries were prepared in
triplicate to determine the robustness of each protocol. The efficiency
of the 3 kits for generating sequencing libraries was compared
qualitatively and quantitatively. DNA quality was controlled by
high-sensitivity capillary electrophoresis (Figure 3a ). The
electrophoregrams obtained on Agilent chip showed negligible amounts of
free adapters in the final DNA libraries after the two SPRI purification
steps whatever the kit used. Then, the number of adapter-ligated
molecules in the libraries was quantified by qPCR Kapa assay using
primers targeting the P5 and P7 sequences of the adapters, that
correspond to the binding sequences to the Illumina flow cell
(Figure 3b ). The ligation step of the kit NxSeq was the most
efficient, leading to a library DNA concentration of 8.2 nM. For this
reason, the NxSeq AmpFREE Low DNA kit was preferred over the two other
kits and was included in the novel SSV-Seq 2.0 method for the PCR-free
sequencing library preparation.
The optimized PCR-free protocol was then tested for the analysis of a
rAAV vector batch, in parallel to the original SSV-Seq technique
(Lecomte et al., 2019). The new protocol was applied to a purified
rAAV8-CAG-GFP vector batch that was produced in HEK293 cells by plasmid
transfection (Figure 1, PCR-free protocol ). The initial amount
of rAAV vectors needed for the analysis and determined by free ITR qPCR
(D’Costa et al., 2016) had to be increased from 2x1011vector genomes for the original SSV-Seq method to
8x1011 vg for the SSV-Seq 2.0. For each protocol, an
additional replicate was used to control the second strand synthesis
step. After DNA extraction and second strand synthesis, 300 ng of
fragmented DNA, as determined by fluorometric quantification, was used
as input for the PCR-free library preparation. The PCR-free library DNA,
devoid of free adapters, was quantified by qPCR Kapa. The concentration
of adapter-ligated fragments was on average 4.5 ± 0.2 nM in a final
volume of 16 µL compared to a mean of 49.8 ± 5.1 nM in a final volume of
30 µL for the PCR-mediated protocol, which is above the minimal
concentration required for Illumina sequencing. In conclusion, our
optimized SSV-Seq 2.0 protocol allows to reach the quality and quantity
of library DNA necessary for the analysis of AAV vectors by HiSeq
Illumina sequencing.