98% of reads have 3' G....WHY?

[grr14]

Hi,

Just completed small RNAseq with TrueSeq library prep and NextSeq500 sequencer.

For whatever reason, even after trimming of the 3' adapter, 98 % of reads have 3' G ending? Supposedly the index sequence should be trimmed with the 3' prime adapter too. Any ideas then for the heck happened?

Thanks in advance,
Greg

[GenoMax]

2-channel SBS technology used in NextSeq: https://www.illumina.com/content/dam...hannel_sbs.pdf

Absence of a signal is being interpreted as a G. How may cycles did you run BTW?

[grr14]

Hi,

How is the sequencing reaction continuing through the 3'adapter if the G indicates no base detected?

[grr14]

We use 50 cycles

[GenoMax]

How many G's are there in your reads at the end (since 98% of the reads have them)?

I am assuming that a cluster that was productive in earlier cycles may be called a G if it stopped producing usable signal in later cycles. If it is only a base or two (of G) you could trim that base off but if you are seeing poly-G's at the end of reads then you may want to check with Illumina tech support.

There is one past thread about this observation: http://seqanswers.com/forums/showthread.php?t=45130

[boffellid]

Having a G after a cluster becomes non-productive would make sense if the sequence stopped (or turned into garbage) after that G, but the reads actually continue into the 3' adapter with high quality (and the G is also high quality). I cannot see how a cluster can become unproductive for only one cycle.

[GenoMax]

That is the reason I asked @grr14 as to how many G's he sees at the end of his reads (single or poly-G). I was thinking of productive clusters turning non-productive late in the sequencing run and staying in that mode, rather than just for one cycle.