This is a test version of Biostars. For the public version, visit https://www.biostars.org.
I have less uniquely mapped reads for my TF, while input has high uniquely mapped reads.

Dears, I have a question regarding Chip-seq, and it would be so nice to know your idea. I have run Chip-seq, I had 40 samples (2 biological replicates from Human cells, 20 input as ctrl and 20 of my low abundant TF) and I run them on Next seq platform, and although in this platform we have the possibility to get up to 60 Gbp of data, I got only 47 Gbp, so my first question is what is the probable reason for this that I have not used the whole capacity of sequencing?

Then I have run quality controls and these things on data, everything looks ok. Then I did mapping using Bowtie2 in Galaxy, and the percentage of uniquely mapped reads for all of the samples are more than 75%, so that is fine as well. But the number of uniquely mapped reads for inputs are around 10 million reads, while for my TF they are about 4- 5 million uniquely reads per sample. while I could see the minimum based on Encode is 10 million. :( if my sequencing approach has already the capacity to sequence more but I did not get more reads, what does it show? Does not it show that my TF did not have more binding sites to be sequenced? What does it show? Could you help me with this and let me know if I should repeat the sequencing then what should I consider before re-running? Thanks in advance for your help and kind consideration, Bests

chip-seq sequencing

These are numbers obtained after mapping. I select the PCR cycles by running a qPCR so I do not think that I have saturated them. I will go through post-processing steps and filterings and will let you know how does it look like. I have quantified the libraries using Qubit and Bioanalyzer and I took 1.6 PM of library pool for sequencing.

That sounds like you simply have uneven loading of libraries, not uncommon. Normalization should take care of it given the quality of the data is ok in terms of number of callable peaks and fraction of reads per peaks (FRiPs).

1 answer

although in this platform we have the possibility to get up to 60 Gbp of data, I got only 47 Gbp, so my first question is what is the probable reason for this that I have not used the whole capacity of sequencing?

Under-clustering, which means you did not load the optimal amount of DNA.

Does not it show that my TF did not have more binding sites to be sequenced?

What does that mean? Are these numbers obtained after all filtering steps? Do you have a lot of PCR duplicates that indicate the saturation for each library might already be reached? Please be more precise. Best would be to present the flagstat results after filtering the BAM file for MAPQ > 20, removing PCR duplicates and keeping only primary alignments. Samtools can do all that filtering, e.g. samtools view -q 20 -f 0 -F 1284 -o filtered.bam your.bam given you marked duplicates before, e.g. using samblaster. is this simply the "raw" result which could mean that libraries have not been properly balanced so some are overrepresented in the pool you loaded while others (such as your TF ones) are underrepresented. That probably comes down to inaccurate quantification. How did you do it, Bioanalyzer, qPCR, if at all? What kind of input, chromatin or IgG? You should perform some library complexity estimates, such as with preseq and then show the results.

Log in to answer this question.