Question: map fasts files
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gravatar for Learner
11 days ago by
Learner 80
Learner 80 wrote:

Hello,

I have a bunch of FASTQ files (10 samples). I try to load them in R for mapping them but I cannot figure out how to do For example, Dseq2 requires bat files and not fastq, so I cannot find the way how to import fastq and analysis

I tried to use Rsubread 1.26.1 but for example after I do the following command I get this error ERROR: the output directory is not writable, but the index builder needs to create temporary files in the current directory. Please change the working directory and rerun the index builder.

I do setwd() to a good place but still I get error

Can someone comment how to start the analysis for Fastq files ?

rna-seq fastq R • 118 views
ADD COMMENTlink written 11 days ago by Learner 80

Why are you trying to use R to do mapping? Isn't that better done on the command line in unix?

ADD REPLYlink written 11 days ago by GenoMax42k

@genomax it does not matter for me I thought it is easy in R :-) but the main reason is because i want to do expression analysis in R, so I thought R is the best way to go . can you give me a solution ? I will try to do it or give me the direction.

ADD REPLYlink written 11 days ago by Learner 80

What aligner(s) do you have access to/have installed on your system? STAR, bbmap, HISAT2 can all be options on the command line. I suppose Rsubread should work too in R but I don't have any experience with it.

ADD REPLYlink modified 11 days ago • written 11 days ago by GenoMax42k

@genomax I have Mac, I believe I can install any of them, which one is more stable ? or recomened more to be used ? because I believe one should have a reference for alignment so I don't know how any of them works. Please advise

ADD REPLYlink written 11 days ago by Learner 80

STAR is quite often used and pretty fast, but requires a lot of RAM (25-30GB) for big genomes. Are you working on human data?

ADD REPLYlink written 11 days ago by WouterDeCoster26k

@WouterDeCoster yes it is human data but I only have 10 samples , do you think that I need such a Ram?

ADD REPLYlink written 11 days ago by Learner 80

Yes, that's what you need for loading the index while aligning a single sample. I don't know about your computer specs, but perhaps HISAT2 can be used then. Or do you have access to a cluster/server for bigger jobs?

Note that you can also use more lightweight pseudo-alignment approaches such as salmon, which nicely work with DESeq2 using tximport, see for example here. These shouldn't be a problem on "slightly less strong" hardware.

ADD REPLYlink modified 11 days ago • written 11 days ago by WouterDeCoster26k

Yep. STAR/bbmap will need ~30G of RAM for human genome.

ADD REPLYlink modified 11 days ago • written 11 days ago by GenoMax42k

@genomax I have a mac with 32 GB of ram or a windows7 cluster with 64 RAM. let me know which one should i use?

ADD REPLYlink modified 11 days ago • written 11 days ago by Learner 80

Try STAR out on your Mac and see if it works. As long as you do one sample at a time it may. You could download pre-made STAR indexes from here.

ADD REPLYlink written 11 days ago by GenoMax42k

@genomax Thanks, I am having problem with this STAR , I downloaded it , then I cd to the source and then I get error like this make STARforMacStatic CXX=/usr/bin/gcc /usr/bin/gcc -c -O3 -pipe -std=c++11 -Wall -Wextra -fopenmp -D'COMPILATION_TIME_PLACE="Mon Feb 12 16:03:45 CST 2018 :/Users/admin/Desktop/STAR-master/source"' -D'COMPILE_FOR_MAC' SharedMemory.cpp clang: error: unsupported option '-fopenmp' make: * [SharedMemory.o] Error 1

ADD REPLYlink written 11 days ago by Learner 80

Get pre-compiled binary for macOS here.

ADD REPLYlink written 11 days ago by GenoMax42k

@genomax I I got both of them, can you please now tell me how to use it ? should their format be .dms right?

ADD REPLYlink written 11 days ago by Learner 80

STAR is the executable. You may need to do sudo chmod a+x /path_to/STAR to make it executable via terminal.

ADD REPLYlink modified 11 days ago • written 11 days ago by GenoMax42k

@genomax which one of these I should download http://labshare.cshl.edu/shares/gingeraslab/www-data/dobin/STAR/STARgenomes/ENSEMBL/homo_sapiens/ENSEMBL.homo_sapiens.release-83/

ADD REPLYlink written 11 days ago by Learner 80

You have to download all of them. These file together form the index set.

ADD REPLYlink written 11 days ago by GenoMax42k

Human RNA-seq using bwa? I wouldn't call that the next best bet tbh.

ADD REPLYlink written 11 days ago by WouterDeCoster26k

I don't pay attention to tags on posts since they don't always go with contents of posts :-)

I will edit my comment in post above.

ADD REPLYlink modified 11 days ago • written 11 days ago by GenoMax42k

Yes, tags are trouble, but this time there was additional evidence ;)

but the main reason is because i want to do expression analysis in R, so I thought R is the best way to go

ADD REPLYlink written 11 days ago by WouterDeCoster26k
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