cluster density problem in TruSeq Small RNA library sequencing

neda · 07-21-2019, 07:31 AM

Dear all
I’ve sent a 8-indecies pooled sample for TruSeq Small RNA library illumina sequencing. Mention that I didn’t use kit and I used CATS protocol to make the library. QC was passed successfully. Concentration was 574ng/µl and 3594 nM. The size was 246 bp. Now my problem is that the company informed me that due to base concentration issue, they can’t calculate cluster density. Sequencing went successfully after 8 cycle. In order to fix this issue, they told me need put phiX more than 50% but they can’t guarantee the result. and I might get the half of the data except the amount of PhiX rate. Now I want to know is that amount of result scientifically validate. Or is there any other way to get a better result?

luc · 07-22-2019, 02:13 PM

Sorry, I do not fully understand your description of the problem. In any case, it would be best to pool your libraries at a low percentage with other (best complex) libraries (that have differing barcodes) to get a look at data of these libraries.

I assume the problem is the poly-A tail added by the CATS protocol? The quality in this region will certainly be low if sequenced straight-up. On the other hand, you should not need data for these cycles of the read.

neda · 07-24-2019, 01:03 AM

by percantage you mean the concentration?

neda · 07-24-2019, 01:10 AM

by percantage you mean the concentration? unfortunately each of 8samples were at least 100 ng/microliter before pooling. I think It's high. do you think if I dilute the sample and resequence it with 50% of PhiX the problem would reded?

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07-21-2019, 07:31 AM	#1
neda Junior Member Location: Iran Join Date: Nov 2017 Posts: 3	cluster density problem in TruSeq Small RNA library sequencing Dear all I’ve sent a 8-indecies pooled sample for TruSeq Small RNA library illumina sequencing. Mention that I didn’t use kit and I used CATS protocol to make the library. QC was passed successfully. Concentration was 574ng/µl and 3594 nM. The size was 246 bp. Now my problem is that the company informed me that due to base concentration issue, they can’t calculate cluster density. Sequencing went successfully after 8 cycle. In order to fix this issue, they told me need put phiX more than 50% but they can’t guarantee the result. and I might get the half of the data except the amount of PhiX rate. Now I want to know is that amount of result scientifically validate. Or is there any other way to get a better result?

07-24-2019, 01:03 AM	#3
neda Junior Member Location: Iran Join Date: Nov 2017 Posts: 3	by percantage you mean the concentration? Last edited by neda; 07-24-2019 at 01:11 AM.

07-22-2019, 02:13 PM	#2
luc Senior Member Location: US Join Date: Dec 2010 Posts: 453	Sorry, I do not fully understand your description of the problem. In any case, it would be best to pool your libraries at a low percentage with other (best complex) libraries (that have differing barcodes) to get a look at data of these libraries. I assume the problem is the poly-A tail added by the CATS protocol? The quality in this region will certainly be low if sequenced straight-up. On the other hand, you should not need data for these cycles of the read.

07-24-2019, 01:10 AM	#4
neda Junior Member Location: Iran Join Date: Nov 2017 Posts: 3	by percantage you mean the concentration? unfortunately each of 8samples were at least 100 ng/microliter before pooling. I think It's high. do you think if I dilute the sample and resequence it with 50% of PhiX the problem would reded?