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Hi, everyone,
I have a question regarding the Miseq RNA sequencing and seek for experienced advice.
I have 18 libraries prepared by Illumina RNA Sample prep V2 kit for RNA seq on Miseq platform. They are 6 samples*3 biological replicates. I am pooling 3 samples per run.
The run #1 and 2, which sequenced the 1st and 2nd biological replicates of the sample A, B and C, have pretty good results: ~800K/mm2 of cluster density. But the 3rd run, sequencing the 3rd biological replicate of the same set of sample, has only ~550 K/mm2. But my colleague made a mistake when normalizing libraries so i personally did a repeat. but the cluster density is still low as about ~550. Then I decided to run another set of sample, the 1st biological replicate of sample D, E and F. This time, the cluster density is about ~800 as run #01 and 02. So i believe this low cluster density is related to these particular samples, not to machine or running reagents. All libraries are validated and quantified without any problem identified.
My question is: can I pool the reads generated from run #03 and 04 (both are sequencing the 3rd biological replicates of sample A, B and C) as 1 set of data for the analysis? I am using CLC Genomics Workbench for the transcriptome analysis using RPKM method. Will this pooling cause an artificals on the results of gene transcription levels or bias for the analysis?
Thank you all for your advices.
I have a question regarding the Miseq RNA sequencing and seek for experienced advice.
I have 18 libraries prepared by Illumina RNA Sample prep V2 kit for RNA seq on Miseq platform. They are 6 samples*3 biological replicates. I am pooling 3 samples per run.
The run #1 and 2, which sequenced the 1st and 2nd biological replicates of the sample A, B and C, have pretty good results: ~800K/mm2 of cluster density. But the 3rd run, sequencing the 3rd biological replicate of the same set of sample, has only ~550 K/mm2. But my colleague made a mistake when normalizing libraries so i personally did a repeat. but the cluster density is still low as about ~550. Then I decided to run another set of sample, the 1st biological replicate of sample D, E and F. This time, the cluster density is about ~800 as run #01 and 02. So i believe this low cluster density is related to these particular samples, not to machine or running reagents. All libraries are validated and quantified without any problem identified.
My question is: can I pool the reads generated from run #03 and 04 (both are sequencing the 3rd biological replicates of sample A, B and C) as 1 set of data for the analysis? I am using CLC Genomics Workbench for the transcriptome analysis using RPKM method. Will this pooling cause an artificals on the results of gene transcription levels or bias for the analysis?
Thank you all for your advices.