Q:
I’m trying to run FunSeq2 through the online application. I’ve tried a few times over the past few days, but always get this error message: "Sorry, but the requested page is unavailable due to a server hiccup." Can you please advise?

I am still not able to run FunSeq2 online; I’m getting the same error message as before. I also downloaded the source scripts and can run example input, however, it is extremely slow when trying to run >1,000 input SNPs (20hr+ on cluster computing). I noticed that Supplemental Table 5 says it can process 2,000 SNPs in 2 minutes – is this for the online version only?

Do you have suggestions for working with the online version? I’ve tried tab delimited .bed and a mix of double spacing for the positions and tabs for the alleles .bed (as suggested by the ARVIN method), and neither work for me.

A:
I have double-checked the webserver and done some testing, the server works as usual. But I noticed some input formatting may cause the error you had. for example, please follow the format we suggested, and use <tab> delimiter not space. If you still have problems, could you share your input file, so we can help to figure out the problem?

However, considering the webserver is based on an old version of FunSeq2, we recommend you use our latest version. We have also prepared a pre-calculated whole-genome score on hg19 and hg38(leftover). You just need to download the score and use tabix tools and bed to query. For details, please refer http://funseq2.gersteinlab.org/downloads

Q:
The Funseq2 Web Server goes down these days. Would it be available in the next few days?

A:
The Funseq2 web server is up and running now. It has some suspicious activity on the server recently and we are keeping on monitoring it.
If you are submitting your own query, please try to use the correct format, or it will shows ‘service unavailable’ service.

As an alternative, you can also download the whole genome annotations for both hg19 and hg38 from funseq3.gersteinlab.org, then use tabix to query.

Q:
I found your paper regarding to Funseq2 and quite interested at how do you assign weight or calculated weight for each category. From weighted scoring schema, I could see different categories have different weight, but I am not sure how do you decide them .

A lot bit about me: I am interested pediatric genetic diseases and working on a birth cohort at Beijing Children Hospital as assistant professor.

A:
It’s an entropy-based scheme in the paper. It’s also described in
various FunSeq lectures (on lectures.gersteinlab.org).

The details of Funseq2 can be found in our paper: https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0480-5. Simply, In Funseq2, we firstly to define a weighted score for each feature based on their distribution of features in random selected common variants. Discrete and continuous features use slightly different way (refer the formula 1 and 2 in the paper).
for a discrete feature, like ‘In sensitive regions’: [see image]

if there are 20 out of 2000000 random common variants are overlapping with sensitive regions, the Pd will be 20/2000000 = 0.0001 , then [see image]
will be used to get the weight for ‘In sensitive regions’

For the continuous feature, it uses:
[see image]

Q:
I have downloaded the Whole genome scores(hg19) both Version 2.1.6 and 2.1.0 in the project website you provided http://funseq2.gersteinlab.org/downloads. There no score when I queried the codign region, but in the Whole Genome Query interface displays the results, such as chr1:11073808-11073808. I will be so appreciated if you could kindly tell me the reason of this problem. Thank you for your kind consideration of this request.

A:
The genome score you downloaded only includes non-coding variants. For coding regions, the score mostly reply on VAT annotation (another tool by our lab: vat.gersteinlab.org). Also whole genome score including both coding and non-coding will be a very large file, which over 50G after compressed. So we provide a query server: http://funseq3.gersteinlab.org/ . Thanks also for pointing out this issue on the download page, and we will update the webpage with clear and detailed file descriptions.

Q:
Recently, I used FunSeq2 to identify non-coding regulatory variations in my
bladder cancer research. In promoter analysis, I discovered the original
file, gencode.v19.promoter.bed, which downloaded from
http://funseq2.gersteinlab.org/static/data_context2.1.2/gencode/, having the
promoter areas of ranking from 1 to 8979 bp, that was inconsistent with the
definition in your article (promoters defined as -2.5 kb from transcription
starting sites).

So, I checked the process script 3.gencode.process.pl, which was downloaded
from http://funseq2.gersteinlab.org/scripts_dev/ and suited for gencode.v16.
This script generated gencode.v16.promoter.bed
(http://funseq2.gersteinlab.org/static/data_context2.1.0/gencode/), and the
BED file’s 3rd column minus 2nd column all equals 2500 (2.5 Kb). After
comparing, I noticed that the latest gencode.v19.promoter.bed has the
additional 5th column, so I realized the 3.gencode.process.pl script had
been re-edited, but I did’t find the latest version on the internet.
Therefore, I wonder whether the latest 3.gencode.process.pl redefined the
meaning of promoter. If it does, can I get one copy of this script?

A:
The promoter file was derived from PCAWG promoter set, which may consider chromHMM segmentation information. Yao have updated this in the v2.1.2, then I keep it in the latest version. User can replace the right file using their own definition of promoters.

The promoter file included in Funseq 2.1.2 is based on PCAWG consortium’s definition, which considers ChromHMM segmentation information. So it will not be exactly 2kb or 2.5kb upstream of TSS.