Question: Can I use a melting curve analysis after the Kapa qPCR protocol to determine the quality of my 10x libraries?
Answer: It is not recommended to run a melting curve analysis after Kapa qPCR quantification on 10x libraries to assess library quality. This is because none of the 10x library types will look like a standard Illumina library and would be classified as having an "abnormal" melt curve.
10X libraries have different insert length distributions:
- SC3' v2, SC3' v3 and SC5' Gene Expression libraries have ~270-280 bp inserts
- 5' VDJ Enrichment library inserts peak at ~490 bp with a tail consisting of shorter fragments
- Genome and CNV libraries have inserts >400 bp
- Single Cell ATAC libraries show periodicity of chromatin structure with most inserts ranging from 30-400bp
- Protein and CRISPR Feature Barcode libraries have a single peak size
- 10X libraries can have unique/specific features associated with the library that can fundamentally change the way the library melts:
- Presence or absence of PolyT/A
- TSO sequence in all SC5' libraries
- Differences in GC content
- The melt curves haven't provided any useful diagnostic information with respect to library quality and the presence or absence of adaptor dimers for 10x libraries.
Quality of the final 10x libraries can be determined via Bioanalyzer, Tapestation of Fragment Analyzer run.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell ATAC, Genome/Exome, Single Cell CNV