Question: Is it possible to perform amplicon sequencing?
Answer: We haven't tested amplicon sequencing and unfortunately cannot provide specific recommendations; however, some considerations in doing this would be:
- Ensure input molecules are longer than 10kb. If molecules are shorter than 10 kb, amplification efficiency may be impacted. If your amplicon of interest is shorter, the input molecule size may be increased by concatenation.
- If the genomic region(s) of interest is small, multiple samples could be assayed by multiplexing the same region from different individual organisms in a single Chromium Genome run. Barcode each individual sample before pooling samples, and sequence with adequate depth to assemble each molecule with its sample barcode.
- Load target regions in carrier DNA to reduce barcode collision (i.e. when a GEM has greater than 1 molecule from the same locus) and to reduce the number of copies of the target region that must be assembled. When loading 1.25 ng DNA, approximately 500 kb is partitioned per GEM and there are approximately 1 million GEMs generated per Chromium run. Remove carrier DNA post-library construction to avoid sequencing off-target DNA.
Note: The Long Ranger pipeline is not designed for amplicon sequencing and therefore downstream analysis will require a custom pipeline.
Alternatively, it would be very straightforward to detect variants within amplicons using our Exome or targeted workflow:
https://support.10xgenomics.com/genome-exome/index/doc/technical-note-target-enrichment-using-custom-baits-with-the-chromium-genome-reagent-kit
Products: Genome, Exome