Low quality in the middle of the read?

[MG1655]

Edit: okay, looking at other threads, I think my problem was also not including the NNNNN diversity in the oligos for read2... thanks! and sorry for the spam.



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Hi all, I recently did a PE100 run and saw that while the fwd read was fine, my reverse read has some quality issues.

I submitted an PCR amplicon library and generated the calibration diversity by encoding NNN's into my primers. I also used custom sequencing primers.

I'm just troubleshooting and wondering why read2 did not work as well, was it because of the custom sequencing primers, or because there's something problematic with the sequence we're targeting? I wold like to learn how to avoid this issue in the future

@HS2:2341242ACXX:6:1101:13025:2685 2:N:0:ATCACG
ACTTCCCTGCTGGCGGTTGCCACGCTGAACGGCGAGNNNNNNNNNNNNNNNNTGNNTGNNGGGGAGATCGTGGATGCAAGGCGCCTGGGGGGGGTGCGTT
+
+1=8A:BDDDDDDDIIEEIIIEIIIDDDIIDI####################################################################

Also, there are some reads that have NNN and ### in the middle of the read but not at the beginning and end, which is somewhat interesting. I thought once the quality hits bottom, it doesn't go back up again.

@HS2:2341242ACXX:6:1101:13880:2694 2:N:0:ATCACG
GCTATGCTGCTGGCGGTTGCCACGCTGAACGGCGAGNNNNNNNNNNNNNNNNTGNNTGNNGGGGAGTTCGTGGATGCACCGCGCCATGGGAGTGATGCGT
+
+1=?DFFFFHHHHHJJGHHJJGJJIJIIIJIJ(.5=################?B##9<##+2++258?CBDBBDACA@CD9<@DDDBCCDCB8?@ACDD<