Directional RNAseq troubleshooting

[deepakpatilp]

Hi Friends

I am new to Illumina sequencing. I am following a published protocol for directional RNA sequencing (http://www.cell.com/molecular-cell/a...765(10)00416-8). Recently I had a run which gave me very less number of clusters. The final fragment size is approximately 150-500 bases. Though I used bioanalyzer to determine fragment size distribution, I am pretty not sure of how to determine the molar concentration of the library. Please help with your suggestions. What could be the reason for getting less clusters? Could it be adapter contamination?

Please help
DEEPAK

[mnkyboy]

I am pretty not sure of how to determine the molar concentration of the library.

How did you try to to determine the molar concentration?

[deepakpatilp]

How did you try to to determine the molar concentration?

I relied on bioanalyzer readings and took an average of the fragment sizes (150+500/2=325 bp)to quantify the library fragment concentration.

[mnkyboy]

I would try measuring mass by picogreen or doing qPCR. The bioanalyzer could just be underestimating your molar amount (which is not unusual).

Also how much did you think you loaded and how many clusters did you get?

You may also have to run a titration flow cell, which sometimes is the only way to know with a non-standard library.