Improved precision, sensitivity, and adaptability of ordered two-template relay cDNA library preparation for RNA sequencing

TABLE 3.

Summary of Sanger sequencing of plasmid-cloned adapter-dimer cDNAs

Buffer 4B Read2:cRead1 junction Primer duplex overhang Primer duplex paired First cDNA nucleotide First cDNA base paired Primer duplex dependent? NTA dependent? Detectable by Read1 sequencing?
+1 Y primer duplex v2 and 3′rC no UMI adapter template C|GAGATCGG +1 C No G 0 No Yes Yes
C|GAGATCGG +1 C No G 0 No Yes Yes
T|GAGATCGG +1 T No G 0 No Yes Yes
C|-AGATCGG +1 C rC* A −1 No Yes Yes
C|-AGATCGG +1 C rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
T|-AGATCGG +1 T rC* A −1 No Yes Yes
C|‐‐‐‐‐‐GG +1 C rG G −6 Yes No No
C|‐‐‐‐‐‐GG +1 C rG G −6 Yes No No
C|‐‐‐‐‐‐GG +1 C rG G −6 Yes No No
C|‐‐‐‐‐‐GG +1 C rG G −6 Yes No No
T|‐‐‐‐‐‐GG +1 T rG* G −6 No Yes No
T|‐‐‐‐‐‐-G +1 T G* G −7 No Yes No
T|‐‐‐‐‐‐-G +1 T G* G −7 No Yes No
T|‐‐‐‐‐‐-G +1 T G* G −7 No Yes No
T|‐‐‐‐‐‐-G +1 T G* G −7 No Yes No
T|‐‐‐‐‐‐-G +1 T G* G −7 No Yes No
T|‐‐‐‐‐‐-G +1 T G* G −7 No Yes No
T|‐‐‐‐‐‐-G +1 T G* G −7 No Yes No
T|‐‐‐‐‐‐-G +1 T G* G −7 No Yes No
+1 Y primer duplex v2 and 3′ddCRN5 UMI adapter template T|GTTTTCTAGATCGG +1 T No G 0 No Yes Yes
C|GCGGCCCAGATCGG +1 C No G 0 No Yes Yes
T|‐‐AGCTTAGATCGG +1 T A A −2 Yes Yes Yes
T|‐‐AACTTAGATCGG +1 T A A −2 Yes Yes Yes
C|‐‐GGCTAAGATCGG +1 C G G −2 Yes Yes Yes
T|‐‐GGGCTAGATCGG +1 T A G −2 Yes Yes Yes
C|‐‐GGTACAGATCGG +1 C G G −2 Yes Yes Yes
T|‐‐GTCATAGATCGG +1 T A G −2 Yes Yes Yes
T|‐‐GCGTAAGATCGG +1 T A G −2 Yes Yes Yes
T|‐‐GAGAAAGATCGG +1 T A G −2 Yes Yes Yes
T|‐‐GGGTTAGATCGG +1 T A G −2 Yes Yes Yes
T|‐‐-GGTTAGATCGG +1T N G −3 Yes Yes Yes

  • In sequences of Read2:cRead1 junction column, the “−” symbol was used to emphasize sequence that was excluded from the junction. In the primer duplex paired column, the “*” symbol was used to emphasize that a mismatch was observed. Column 7 defines whether the adapter dimer was dependent on a correct base pair between the primer duplex +1 nt and the 3′ nt of adapter template. Column 8 defines whether the adapter dimer was dependent on an NTA to the +1 primer duplex to template jump to the adapter template. Column 9 defines whether the adapter-dimer cDNA could be sequenced on an Illumina instrument, as assessed by the presence of a complete cRead1 primer sequence.

This Article

  1. Published in Advance December 3, 2024, doi: 10.1261/rna.080318.124 RNA 31: 224-244

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