w2rap-contiger with 150bp pe library about w2rap-contigger

[bjclavijo]

Hi Stephan. That is not a problem with read length, but rather your qualities having incorrect values (or extreme ones).
Which kind of files are you using as input files? Fastq? can you paste the command line you used too? I would recommend running fastqc or something like that to get an idea how your files look like.

[StephanHolgerD]

Hey,
I provided gzipped fastq files as input. But I checked the quality values with fastqc and i also trimmed my reads using trimmomatic, before I try to start W2rap-contiger.

here is my starting commandline

:~$ '/home/test/Programme/w2rap-contigger/bin/w2rap-contigger' -o d1131_cat_30_nohead_130 -p d1131_cat_30_nohead_130 -r '/media/test/daten/Stephan/Genom/02_processed_Data/d1131_cat_30_minlen130/d1131_cat_30_nohead_130_1P.fq.gz','/media/test/daten/Stephan/Genom/02_processed_Data/d1131_cat_30_minlen130/d1131_cat_30_nohead_130_2P.fq.gz' -K 80 -t 40

[Vickywzy]

Hey,
I also have this error , can you tell me how to solve this error?

[jonwright99]

Can you send me the command line you're using to start w2rap and paste in a few lines of your FASTQ files?

[kokyriakidis]

@jonwright99 @bjclavijo Does w2rap-contiger support gz files as input? How do I use multiple fastq.gz files as input? Just adding multiple times the -r parameter?

[jonwright99]

We've not added functionality to use gzipped files yet so your FASTQs must be unzipped for now. For multiple files use a single -r parameter like so; -r file1.fastq,file2.fastq,file3.fastq,file4.fastq

[kokyriakidis]

@jonwright99

1. So, I have to interleave them? There are 3 libraries and are in _1.fastq _2.fastq format.
2. The reads are 100bp and the insert size of the libraries are i) 240bp ii) 420bp iii) 800bp. Do you recommend using all of them? Or should I exclude the 240 one?

[jonwright99]

w2rap has no concept of separate libraries as yet (another improvement we hope to make in the future). We generally sequence one library with 600-700bp insert size to around 45x coverage with 250bp reads. For your data I would include the largest libraries until you have required coverage. 100bp reads are pretty short so depending on the complexity of your genome, w2rap may struggle to build the graph.

[kokyriakidis]

@jonwright99

1) So, should I interleave each of the 2 libraries (420bp and 800bp) and put them in the program as file1.fastq (420bp) and file2.fastq(800bp)? Or should I concatenate the fastq files (420bp and 800bp) and input file1_R1.fastq and file1_R2.fastq?

2) This is the k-mer histogram (using KAT) from the 2 libraries (420bp and 800bp).

This is the k-mer histogram (using KAT) from the 3 libraries (240bp, 420bp and 800bp).

If I use only the 420bp and the 800bp libraries, should I set min frequency =5 at the w2rap contiger?
If I use the 240bp, 420bp and the 800bp libraries, should I set min frequency =9 at the w2rap contiger?

3) According to the FASTQC statistics i calculated that :

i) With 420bp and the 800bp insert libraries I have 29x genome coverage
ii) With 240bp, 420bp and 800bp insert libraries I have 50x genome coverage

How should I proceed? Do I use eventually all the libraries? Where to cut the min frequency?

Thanks for your help!

[jonwright99]

Your coverage is pretty low using just the largest 2 libraries so I think you should include the 240bp one as well. You will need to set the min frequency to 9 in this case. You can add the files like so;

-r 240_r1.fastq,240_r2.fastq,420_r1.fastq,420_r2.fastq,800_r1.fastq,800_r2.fastq

The file order shouldn't matter as once loaded the paired reads are connected by their read id. You could alternatively concatenate all the r1 files into 1 FASTQ and all the r2 files into another then load them.

Let me know how you get on.

[kokyriakidis]

@jonwright99 Happy new year!
I am trying to do the LMP Processing but when I try to run lmp_processing <read_file_list> it throws me errors like:

 File "w2rap/lmp_processing", line 100
    print "\n#### w2rap LMP processing ####\n"
                                             ^
SyntaxError: Missing parentheses in call to 'print'. Did you mean print("\n#### w2rap LMP processing ####\n")?

But this is not the only one. When I edit the script and put parenthesis in every print command, it throws me other errors! Do you know what is happening?

EDIT:
It turns out that I was using python 3.6 instead of python 2.7, and the syntax is different. Maybe you have to mention it in the guide so that others not face the same issue.

[bjclavijo]

That script is python2 and you're probably using python3? Best, bj

…

On Tue, 1 Jan 2019, 10:14 Konstantinos Kyriakidis ***@***.*** wrote: @jonwright99 <https://github.com/jonwright99> Happy new year! I am trying to do the LMP Processing but when I try to run lmp_processing <read_file_list> it throws me errors like: File "w2rap/lmp_processing", line 100 print "\n#### w2rap LMP processing ####\n" ^ SyntaxError: Missing parentheses in call to 'print'. Did you mean print("\n#### w2rap LMP processing ####\n")? But this is not the only one. When I edit the script and put parenthesis in every print command, it throws me other errors! Do you know what is happening? — You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub <#24 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AAsIDbVOxpzKlFlTiBCtCoUDrsqIi4UNks5u-19IgaJpZM4NPUda> .

[jonwright99]

Thanks for the feedback. I've added a note to the README to say the LMP processing script is written in Python 2.7.

The s_prepare, s_map and s_scaff binaries need to be compiled from source. There is a Makefile in w2rap/deps/soap_scaffolder which should do the trick (I should also add this into the README)

[kokyriakidis]

@jonwright99 I deleted my comment on s_prepare, s_map and s_scaff cause I realized that I had to build them from source after searching all the folders. So you could also add it in the README for future reference.

There is also a folder named cxxopts @ 0b76869. Do I use it somewhere?

Do you use GapCloser module from Soapdenovo or SOAP_n_collapser.py and SOAP_n_remapper.py do the same thing?

[bjclavijo]

Closing here as the remaining questions are for w2rap pipeline now.