caleblareau / asap_to_kite Goto Github PK

Hi there,

Thank you so much for your code. I am going to use Kallisto for downstream analysis of my ASAPSeq data. I could run your code. However after running Kallisto, I can see only few lines in my output file. (Something like not a good matching is happening in my data).

My first assumption is about reverse complimenting issue of my whitelist.

So I have some questions:

1- can I ask what requirements I should check before running your code?
2- How I can tell my code "NOT do reverse complimenting"? ( I used the option -r) but it looks I am doing it in wring way.
3- Have you ever used your reformatted fastq files for using Kallisto? Do I need to use an extra code after using your code before using Kallisto?

I really appreciate any information. Any information, tutorial, readme file would be helpful.

Thank you so much.

Regards,
Sara

Hi there,

Thank you for sharing your new tool. I am trying to run it my Macbook. I have Python3.8.
However I have some issues when running the code.

I appreciate if you provide me the answer:
1- which python version you are suggesting me to use?
2- I get an error on this command:
"opts.add_option("--nreads", '-n', default = 1000000, help="Maximum number of reads to process in one iteration. Decrease this if in a low memory environment (e.g. laptop). Default = 10,000,000.")"

And the error is:
Traceback (most recent call last):
File "", line 1, in
File "/Library/Frameworks/Python.framework/Versions/3.8/lib/python3.8/optparse.py", line 1008, in add_option
self._check_conflict(option)
File "/Library/Frameworks/Python.framework/Versions/3.8/lib/python3.8/optparse.py", line 980, in _check_conflict
raise OptionConflictError(
optparse.OptionConflictError: option -n/--nreads: conflicting option string(s): -n, --nreads

Do you have any solution for me? Appreciate that.

Thanks,
Sohani

I'm trying to run the asap_to_kite_v2.py script on scADT-seq data only. After running bcl2fastq, I only have read1 and read2, but see references to a read3 in the code that is causing me some errors. Is this read3 error because I only have scADT-seq data instead of ASAP-seq? How can I adapt the code for TotalSeqA in asap_to_kite_v1 function?

listRead1 = trio[0]; listRead2 = trio[1]; # listRead3 = trio[2]

title1 = listRead1[0]; sequence1 = listRead1[1]; quality1 = listRead1[2]
title2 = listRead2[0]; sequence2 = listRead2[1]; quality2 = listRead2[2]
# title3 = listRead3[0]; sequence3 = listRead3[1]; quality3 = listRead3[2]

# Recombine attributes based on conjugation logic
if(conjugation == "TotalSeqA"):
    new_sequence1 = sequence2 + sequence1[0:10]
    # new_sequence2 = sequence3
    
    new_quality1 = quality2 + quality1[0:10]
    # new_quality2 = quality 3

out_fq1 = formatRead(title1, new_sequence1, new_quality1)
out_fq2 = formatRead(title2, new_sequence2, new_quality2)

If I comment out all references to a read3, there is the issue with out_fq2 requiring new_sequence2 which typically includes the use of a read3.

I am able to run asap_kite_v2.py but it is generating an error (see below) when trying with the latest version. I am wondering if you could tell what step is going off here?
[sod4003@node105 asap_to_kite]$ python asap_to_kite_A_B.py -f ADT_A -s ADT_A -o out_test

Traceback (most recent call last):

File "/athena/namlab/scratch/sod4003/asap_to_kite/asap_to_kite_A_B.py", line 43, in

A_barcodes = [line.rstrip('\n') for line in open(options.TSA)]

TypeError: expected str, bytes or os.PathLike object, not NoneType

[sod4003@node105 asap_to_kite]$ python asap_to_kite_A_B.py -f test/data1 -s test1 -o one_one

Traceback (most recent call last):

File "/athena/namlab/scratch/sod4003/asap_to_kite/asap_to_kite_A_B.py", line 43, in

A_barcodes = [line.rstrip('\n') for line in open(options.TSA)]

TypeError: expected str, bytes or os.PathLike object, not NoneType

Best regards,

Sohani

Hi,
I applied this script to our ASAP-Seq experiment followed by a kb script. In this experiment, we wanted to use the ADT for separating different samples. The amount of cells recovered by this approach looks fine. However, many of the cells have very low read counts, some of them none at all for the used Antibodies.
I am now trying to figure out, whether this is due to the experimental, or the computational setup. Is it possible to share your script / commands to extract the AB counts via kallisto / bustools?
Do you have any other suggestions to debug this issue?
Thanks for your help and best,
Felix