Hi,
We produced a test ATAC-Seq data with 26.8 million 87bp paired-end reads using Chinese bulbul's feathers. I used Bowtie2 for the alignment and ATACseqQC for the quality analysis. The below are (1) fragment size distribution and (2) library complexity produced by ATACseqQC. I think that we have to optimize the ratio of cell number and Tn5 enzyme concentration. However, I don't know whether I have to increase or decrease Tn5 enzyme concentration. Also, does the library complexity result means that we have to increase the sequencing depth? Any suggestions are very welcome.
Best,
Gary
Hello Gary,
I am one of the co-developer of the ATACseqQC package. Your library fragment size plot is not optimal. It lacks fragments less than 100bp, which should be from OPEN chromatin regions. On the other hand your plots did not show a apparent ladder pattern of mono-,bi-, tri-,...nucleosomes, which suggests overtransposition occurred in your experiment. Two important questions are that 1) did you remove the duplicates? 2) did you have some size selection intentionally or unintentionally, such as Ampure magnet beads or DNA purification columns, which remove fragments smaller than 100bp? Personally, I don't suggest to do more sequencing with your current libraries. It will not give you information of location of completely open chromatin regions.
Haibo Liu
Dear Haibo,
Thank you for your very useful information. I have not removed duplicated reads in the fragment size distribution and library complexity figures.
We didn't make a size selection but used a Qiagen MinElute PCR Purification Kit for the purification as suggested by Buenrostro, et al. (2015). Do you think that the Qiagen kit removes our fragments smaller than 100bp? We only used two feather follicles for this pilot experiment. The number of cells could be very limited. Next time, we may use five feather follicles. Do you think that we should follow a low-cell number protocol without the Qiagen MinElute purification below (Buenrostro, et al. 2013)? Many thanks.
Low–cell number protocol. To prepare the 500- and 5,000-cell reactions, we used the same protocol with some notable exceptions: the transposition reaction was done in a 5µL instead of 50µL reaction. Also, we eliminated the Qiagen MinElute purification before PCR and instead took the 5µL reaction immediately after transposition directly into the 50µL PCR.
Reference
Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 2013;10(12):1213-8. Buenrostro JD, Wu B, Chang HY, Greenleaf WJ. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Current protocols in molecular biology / edited by Frederick M Ausubel [et al] 2015;109:21.9.1-9.
Best,
Gary