who can help me?

[dalin]

the length of high throughput sequencing library is 250bp.

the sequencing quality from the P5 end is OK,but the sequencing quality from the P5 end is very bad.

sorry i don't know how to send the image .

[dalin]

sequencing quality from the P5 end

[dalin]

sequencing quality from the P7 end

[woodydon]

What's your question?

[dalin]

What's your question?

why the sequencing results from P5 end and P7 end are different? Because the results are form same library.

[GW_OK]

What kind of sequencing library? What was the cluster density?

[dalin]

What kind of sequencing library? What was the cluster density?

Thanks for your reply. The wired thing I don’t understand is that, we are using a 250 bp library for PE100 sequencing. While the Q30 of p5 looks good, the P7 Q30 is really bad (except the first base). Can anyone give me some input/suggestion about this. How this happens and how to avoid this. Thanks a lot!

[dalin]

this is the sequence of the library(from T-A clone)
(P5)NNNNNNNNNNNNNNNNNNNN------GTCGGAGAATTCCTTACTAGTAGAACTCTGTTCTTGAGCTAGCATCGATGCTAGCTCAAGAACAGAGTTCTACTAGTAAGGAATTCTCCGAC----NNNNNNNNNNNNNNNNNNNN(P7)

[weigrc]

If the libraries are ~380 bp, the sequencing quality issue should be caused by cluster over saturation.

What are the values of Cluster Density (K/mm2) and Cluster PF (%)???

Though Read 1 and Read 2 are sequencing the same library, but Read 2 clusters re-formation will be affected a lot if too many libraries were loaded...

thanks,
Wei

[GW_OK]

You can absolutely have a great R1 and and a terrible R2 due to overclustering, as weigrc said. That's why we keep asking you about the cluster density.
It also looks like your library is a not-very-diverse amplicon. That can also kill R2, even with normal clustering. You should probably decrease the amount of library loaded.

[dalin]

If the libraries are ~380 bp, the sequencing quality issue should be caused by cluster over saturation.

What are the values of Cluster Density (K/mm2) and Cluster PF (%)???

Though Read 1 and Read 2 are sequencing the same library, but Read 2 clusters re-formation will be affected a lot if too many libraries were loaded...

thanks,
Wei

Thank you so much!I will check the values of Cluster Density !

[dalin]

You can absolutely have a great R1 and and a terrible R2 due to overclustering, as weigrc said. That's why we keep asking you about the cluster density.
It also looks like your library is a not-very-diverse amplicon. That can also kill R2, even with normal clustering. You should probably decrease the amount of library loaded.

thanks,I will upload the date of the cluster density.
thanks for your reply， which explained a lot. Yes I am using amplicon. The worse is that my library has only about 20bp diverse sequence in 5 and 3 end. the sequence in between is a linker， means there is no diverse at all. i cannot change my library. but hope there will be a trick to circle this problem.

[dalin]

If the libraries are ~380 bp, the sequencing quality issue should be caused by cluster over saturation.

What are the values of Cluster Density (K/mm2) and Cluster PF (%)???

Though Read 1 and Read 2 are sequencing the same library, but Read 2 clusters re-formation will be affected a lot if too many libraries were loaded...

thanks,
Wei

thanks for your reply， which explained a lot. Yes I am using amplicon. The worse is that my library has only about 20bp diverse sequence in 5 and 3 end. the sequence in between is a linker， means there is no diverse at all. i cannot change my library. but hope there will be a trick to circle this problem.

[dalin]

What kind of sequencing library? What was the cluster density?

Thanks for your reply.
I'm sorry i don't know the cluster density of the library. The Sequencing company have no information for the cluster density.

[nucacidhunter]

I wonder if you are using custom sequencing primers. Your read2 sequencing primer might not be optimal in addition to other reasons given previously. I think you need to provide more information about the library and sequencing metrics to enable others to help you effectively.