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Hello,
Recently I had a sequencing run on Miseq with custom Read1 and Read2 primers and I’m not very happy with quality of basecalls.
Miseq generated ~700K cluster/mm^2 but Read1 >=Q30 was 88.2% and Read2 >=Q30 71.5%
Could it be the case of not efficient annealing of primers?
Tm of my custom primers:
Read1 77.6°C;
Read2 79.3°C.
Native illumina’s primers have following Tm’s:
Multiplexing Read 1 Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 77.5°C
Multiplexing Read 2 Sequencing Primer
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC 79.2°C
Multiplexing Index Read Sequencing Primer
5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC 79.2°C
My custom primers have almost the same temp. properties.
Do you guys have any ideas or recommendations how custom primers should be designed?
Thank you very much!
Recently I had a sequencing run on Miseq with custom Read1 and Read2 primers and I’m not very happy with quality of basecalls.
Miseq generated ~700K cluster/mm^2 but Read1 >=Q30 was 88.2% and Read2 >=Q30 71.5%
Could it be the case of not efficient annealing of primers?
Tm of my custom primers:
Read1 77.6°C;
Read2 79.3°C.
Native illumina’s primers have following Tm’s:
Multiplexing Read 1 Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 77.5°C
Multiplexing Read 2 Sequencing Primer
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC 79.2°C
Multiplexing Index Read Sequencing Primer
5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC 79.2°C
My custom primers have almost the same temp. properties.
Do you guys have any ideas or recommendations how custom primers should be designed?
Thank you very much!
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