We used some methods very similar to what had been described in Tsai's 2015 GUIDE-seq Nature paper: break DNA, insert barcode, ligate together, PCR amplification. The major if not only difference is that we used radioactivity instead of CRISPR-cas9 to generate DNA double strand breaks. 

I tried the R bioconductor package "GUIDEseq" and the python GUIDE-seq package, and have questions about both methods:

* the R GUIDEseq package: successfully installed, but it requires .bed & .bam files to start with, while all we have are .fastq raw dump from HiSeq.

* the python GUIDE-seq: successfully installed, but when running cutdapt.py, I got the following error:

------------------------------ START -----------------------------------

Traceback (most recent call last):

  File "cutadapt.py", line 61, in <module>

    from cutadapt import check_importability

  File "/ark/home/mx010/.local/lib/python2.7/site-packages/cutadapt/scripts/cutadapt.py", line 61, in <module>

    from cutadapt import check_importability

ImportError: cannot import name check_importability

------------------------------ END -----------------------------------

What should I do? What are the functional differences between the R GUIDEseq package and the python GUIDE-seq? It seems that they are doing different things.

Thanks!

-- Mo

Step1. Bin barcode
./binReads.sh fastqFolder barcodes 1 8 16 p7.index p5.index usedBarcodes
where fastqFolder contains the fastq files and barcodes is the barcode index. 

Q: what is "barcodes" and what is "usedBarcodes"?

Are they file names? If so, how do they look like inside?

Thanks!

Hi Julie,

Thanks for the clarification.

The link for the gzip file http://mccb.umassmed.edu/GUIDEseq/barcodes.bowtie1.index.tar.gz is not valid.

Thanks again,

Mousheng Xu

Hi Julie,

Got the gz package and unzipped it. It contains multiple files. So, still, how to run binReads.sh?

./binReads.sh fastqFolder barcodes 1 8 16 p7.index p5.index usedBarcodes

What should be in the place of "barcodes" and what do 1 8 16 mean?

Thank you!

-- Mousheng 

Mousheng,

If you are following standard protocol of GUIDE-seq, you will not need to change these parameters.  Please look at binReads.sh file to see the parameters and what programs you need.

16 means 16bp barcodes (8 for p7 index and 8 for p5 index)

8 means (mapping with 8 threads)

1  allowing 1 mismatch.

The input fastq file should look like the one at http://mccb.umassmed.edu/GUIDE-seq/testGetUmi/testGetUmi.fastq

Please note that you need to have bowtie1 and R installed for this step, bowtie2 installed for mapping to the genome.  If you are running batch job for Platform LSF, you do not need to modify the script. Otherwise, please make sure to change the "module load" command in binReads.sh accordingly.  

Best regards,

Julie