I followed the ATACseqQC guide and ran the following:

tsse <- TSSEscore(gal1, txs)
summary(tsse$TSS.enrichment.score)

This outputted:

##     Min.   1st Qu.   Median  Mean  3rd Qu.   Max.   NA's
##   0.0000    0.9152   2.3269   Inf   9.6348    Inf    206

The Encode ATAC-seq Data Standards define the TSS enrichment score to be the center of the normalized distribution-- does this refer to the median value in the summary? The official Encode definition is:

Transcription Start Site (TSS) Enrichment Score - The TSS enrichment calculation is a signal to noise calculation. The reads around a reference set of TSSs are collected to form an aggregate distribution of reads centered on the TSSs and extending to 1000 bp in either direction (for a total of 2000bp). This distribution is then normalized by taking the average read depth in the 100 bps at each of the end flanks of the distribution (for a total of 200bp of averaged data) and calculating a fold change at each position over that average read depth. This means that the flanks should start at 1, and if there is high read signal at transcription start sites (highly open regions of the genome) there should be an increase in signal up to a peak in the middle. We take the signal value at the center of the distribution after this normalization as our TSS enrichment metric. Used to evaluate ATAC-seq.

Then my median of 2.3269 is bad, according to their current standards:

<5: Concerning

5-7: Acceptable

>7: Ideal

I'm confused, because even in the example in the ATACseqQC guide, their output is:

##    Min.   1st Qu.   Median   Mean   3rd Qu.   Max.   NA's 
##   0.000    2.227    4.856     Inf    16.722    Inf   3750

where the median is also in the "Concerning" range.

Am I misunderstanding? Should I be looking at the 3rd. Quartile value instead? If not, is there any way to raise the TSS enrichment score? Thank you!

Hi Arpi,

Thank you for reporting this error. I was confused in understanding the definition of TSSE score. I updated the formula for TSSE score calculation. In the development version (1.13.5), I fixed the TSSE score by

TSS scores = the depth of TSS (each 100bp window within 1000 bp each side) / the depth of end flanks (100bp each end).

TSSE score = max(mean(TSS score in each window))

You can change the size for window, step and flanks. By default, all of them are set as 100.

To install the development version of ATACseqQC, please try:

BiocManager::install("jianhong/ATACseqQC")

The TSSE score can be accessed via

tsse <- TSSEscore(gal1, txs)
tsse$TSSEscore

I apologize for the confusing I introduced in TSSE score. Let me know if you have any question in running the new function.

jianhong.

Hi Arpi,

Thanks for the great question! We will output the center of the distribution.

The main developer Dr. Jianhong Ou will update the code so that the output will not contain Inf or NA. He will keep you posted.

Best regards,

Julie