I am currently trying to create an RB-TnSeq library for a Pantoea species. The paper I am basing this off of is https://elifesciences.org/articles/37072#s4.

I have been using it as the protocol is incredibly detailed, and due to the authors detail I feel I have been able to reproduce the methods well for library preparation.

After library preparation we ran the samples on a bioanalyzer and the size was predicted to be the same size (385bp) as in the above paper. There was also no primer dimer called. We also performed pippin prep prior to remove the chance of primer dimer as well (which would have been about 150 bp). However, when I sequence the sample the run was quite strange, appearing to only be 90 bases in each cycle. When we look at the reads we go straight from the transposon enrichment primer (N_spacer_barseq_himar) to the index sequence/ reverse primer. To me that makes me think it could be primer dimer, but I am confused about how this could happen given that we performed pippin prep to remove it or why the size would not be larger in that case.

I have attached a screen shot in case this is helpful of the MiSeq run, as well as a RB-TnSeq diagram from Hentchel et al., 2019 to show what the reads are supposed to look like.

We are currently wondering if there is something in the set up of the run for RB-TnSeq samples that we have that could be causing this?

Any advice about sequencing RB-TnSeq libraries is encouraged, as we have experience in sequencing other libraries but this is our first RB-TnSeq library. Alternatively if anyone has seen a pattern like this before in a MiSeq run and is able to identify it, that would also be incredibly useful.