Probability of sequencing low abundant transcripts from non-normalized library

go9kata · 03-08-2011, 02:57 AM

Dear users,

I am new to this community and this is my first thread, I am also new to next generation sequencing analysis but I have interesting issue and I want to get some feedback from you. Thanks in advance for any replies!

Here is a scenario:
> non-normalized eukaryotic transcriptome library
> 3' Tag Sequencing; Reads of 100nts length; Illumina HiSeq 2000 Single Reads Machine

Two sequencing Attempts:
1) 50 Million Reads at one run with starting RNA consentration X
2) 3 Runs with respectively 10MilReads, 20MilReads, 20 MilReads with starting RNA concentration X

The RNA concentration is the same in the sample before any of the four runs!

Question: Do I get the same set of transcriptome representation between attempt 1 and attempt 2?

Note: Assuming that the probability of getting a very low abundant transcript is constant between runs, i would expect that the chance of observing these transcript in a smaller sampling size is less than in a higher sampling size

Predicted observation: In the second attempt after adding results I would expect that the transcriptome is overwhelmed only by transcripts of higher concentration in the sample, while I would expect to get more less abundant transcrips in the one time sequencing run with 50MilReads (attempt 1)

Thank you for your discussions!

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03-08-2011, 02:57 AM	#1
go9kata Junior Member Location: Frankfurt am Main, Germany Join Date: May 2010 Posts: 2	Probability of sequencing low abundant transcripts from non-normalized library Dear users, I am new to this community and this is my first thread, I am also new to next generation sequencing analysis but I have interesting issue and I want to get some feedback from you. Thanks in advance for any replies! Here is a scenario: > non-normalized eukaryotic transcriptome library > 3' Tag Sequencing; Reads of 100nts length; Illumina HiSeq 2000 Single Reads Machine Two sequencing Attempts: 1) 50 Million Reads at one run with starting RNA consentration X 2) 3 Runs with respectively 10MilReads, 20MilReads, 20 MilReads with starting RNA concentration X The RNA concentration is the same in the sample before any of the four runs! Question: Do I get the same set of transcriptome representation between attempt 1 and attempt 2? Note: Assuming that the probability of getting a very low abundant transcript is constant between runs, i would expect that the chance of observing these transcript in a smaller sampling size is less than in a higher sampling size Predicted observation: In the second attempt after adding results I would expect that the transcriptome is overwhelmed only by transcripts of higher concentration in the sample, while I would expect to get more less abundant transcrips in the one time sequencing run with 50MilReads (attempt 1) Thank you for your discussions!