I searched in the forum and did not find the answer to my question, maybe you can help me.
I’m sequencing my samples for both 16S and 18S, but they come from a vertebrate, so there was a lot of host sequences in our dataset and we are working now on a blocking primer to decrease this abundance.
We are developed a blocking primer specific to the sequence more similar to our host, according to NCBI, and compared to the results when no blocking primer was used.
This is the quality plot without blocking primer:
Additionally, I also compared the total amount of reads per sequencing run (without x with block primer). Without blocking primer:
and with blocking primer:
(I had more samples in the sequencing run with blocking primer, but the samples that were sequenced when no blocking primer was applied were again tested with the blocking primer). The difference in the amount of sequences in “input” and “non-chimeric” is clearly big. I believe this is mostly due to the host sequences that were blocked, however I’ll count them and compare in the datasets without and with blocking primer to see if the numbers are similar.
Here comes my second observation. The abundance of other taxons (such as SAR, Fungi etc) also decreased when the blocking primer was used, which, theoretically, should not happen. Here are two plots only for visualization: without blocking primer, SAR plot
and with blocking primer, SAR plot
So now I’m not sure if the use of blocking primer also influences negatively the general quality of my dataset and to which extension it is a good option or not. I’m having problems to define this threshold and it would be good if someone knew if there are other options to check the efficiency of the primer.
Thanks a lot in advance!!!