It is important that this PCR step does not introduce mutations or bias to the library.
FidelityWhen the sequencing process has a lower overall error rate, less sequence coverage is required, saving expense and time. Also, the exquisite sensitivity of NGS enables the detection of mutations present in samples, such as genomic DNA isolated from cancer tissues. Since such mutations can be present at low levels, it is important that mutations not be introduced during the library preparation process. The use of a high-fidelity DNA polymerase in library preparation results in more accurate sequencing data.
GC BiasGC bias can lead to uneven representation of some sequences within a NGS library.
This in turn can lead to
- Missing regions from assembled sequence
- Inaccurate sequence due to low coverage in certain areas
- Problems with sequence assembly due to uneven representation, including over-representation, of different sequence segments
Several published reports have highlighted the GC bias of some polymerases in amplification of NGS libraries, leading especially to reduced representation of high-GC content regions such as 5 ́ untranslated regions (5 ́ UTRs) and CpG islands.
Quail et al. (2011). Nature Methods 9(1):10-1
Oyola et al. (2012). BMC Genomics 13:1 doi:10.1186/1471-2164-13-1
Dabney and Meyer (2012). BioTechniques, Vol. 52, No. 2, February 2012, pp. 87–94