Question: Is it expected to see low abundance of Ts in cycles 1 and 10 of Read 1 when sequencing 5’ v3 pools?
Answer: Yes, it is expected for 5’ v3 pools. For 5’ v3, Ts at position 1 and/or 10 violate 10x Barcode design requirements (e.g., including homopolymers). For 3’ v4, the 10x Barcodes are base balanced and therefore, do not exhibit this phenotype. Below are Data by Cycle Plots from a 5’ v3 pool on the NovaSeq 6,000 (top) and MiSeq (bottom).
The sequencing quality and Valid Barcodes remains high when sequencing 5’ v3 pools on NovaSeq 6,000 (patterned) and MiSeq (non-patterned) instruments.
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