Cross-contamination risk
During the metabarcoding PCR (here specified as the PCR in which the
metabarcoding marker is targeted), relatively short DNA sequences
(typically <300 bp) are enriched through amplification.
Especially when targeting trace amounts of DNA, PCR amplification can be
highly susceptible to contamination and thereby to false positives. The
risk of contamination when preparing metabarcoding PCRs is the same no
matter which of the three overall metabarcoding approaches is used.
Moreover, regardless of the metabarcoding strategy employed,
cross-contamination can happen between nucleotide tagged and indexed
primer stocks (which are delivered at very high molarity). The risk of
this happening will be similar between the strategies and will depend on
the number of samples and the chosen setup within the employed strategy.
In the following, rather than discussing primer contamination, we will
focus on how the three main metabarcoding approaches differ in risk of
cross-contamination between PCR products after the metabarcoding PCR.
In the one-step PCR approach (Fig. 2B) and the tagged PCR approach (Fig.
2D), PCR products are labelled during the metabarcoding PCR
amplification. In the one-step PCR approach, the metabarcoding PCR is
carried out with primers that target the selected marker and carry both
sequencing adapters and indices. This way, the indexed PCR products can
be immediately sequenced following this one PCR step (Fig. 2B). If the
indexed ready-to-sequence libraries are to be pooled into one pool
before sequencing, then cross-contamination between indexed amplicon
libraries is obviously not of concern. However, if more sequencing pools
are made in which the same index combinations occur across multiple
samples, then cross-contamination between the sequencing pools can be an
issue. A solution is to process them in separate sequencing run batches
to avoid cross-contamination. In the tagged PCR approach, amplicons will
be 5’ nucleotide tagged following the metabarcoding PCR, which means
that cross-contamination between tagged PCR products is not of concern.
However, until the amplicon pools are indexed during library preparation
there is a risk of cross-contamination between amplicon pools if the
same tag combinations are used in different amplicon pools
(Schnell et al.2015). Some laboratories do not reuse tag-primer combinations to
further reduce contamination risk
(see Murrayet al. 2015).
In the two-step approach, sample-specific labelling is not carried out
during the metabarcoding PCR. This creates a risk of cross-contamination
between unlabelled PCR products when handling them prior to the second
PCR (Zizka et al.2019). Therefore, this metabarcoding approach has the greatest
theoretical risk of cross-contamination between PCR amplicons (Fig. 2C).
It is worth mentioning that a few studies adopt modifications of the
two-step approach that eliminates this kind of cross-contamination. One
is to include nucleotide labelling in the first PCR, see Kitson et al.
(2018), and the
other is to carry out both of the two PCRs, i.e. to include both two
primer sets, in the same reaction, see for example Clarke et al.
(2014a).
Irrespective of the chosen approach, cross-contamination can be detected
and filtered out by including sample replicates, PCR replicates, and
positive and negative controls. Thus, these should be included in the
laboratory workflow and sequencing
(e.g. Bistaet al. 2017). An important measure that enables one to filter
out potential contamination during data processing is to use different
nucleotide tag and/or library index combinations on each sample’s
individual PCR replicates as this will allow for restrictive sequence
processing across each sample’s PCR replicates
(Alberdi et al.2018).