For RNA seq data analysis

Question

[bandanaschapagain]

Hi all, I need to identify a software to be used for aligning my reads to the reference genome and getting out the reads that do not align to the reference genome. I have no idea for the softwares available. I hope I can get help. Regards, Bandana

[Amitm]

Have a look here -

Current state-of-the-art for RNA-Seq gene / transcript expression quantification OR just the tools of preference

and specifically for getting reads that do not align, use the --outReadsUnmapped parameter if you use STAR.

[bandanaschapagain]

Thank you for providing such useful link.

[bandanaschapagain]

I am using the below command for STAR. Two surprising things for me are: 1.Time taken by the STAR for read aligment 2. I am not getting the unmapped reads even by using --outReadsUnmapped. I am confused if there something I need to modify for my command here.

../STAR  --genomeDir /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/BMV_002026_in --readFilesIn /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/RNA_Virus_Josef/reads/BMV/JB-BMV_S3_L004_R1_001.fastq  /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/RNA_Virus_Josef/reads/BMV/JB-BMV_S3_L004_R2_001.fastq  --outReadsUnmapped JB_BMV_unmap.out --outFileNamePrefix JB_L004.out

Please help me with this. Regards, Bandana

[Amitm]

Thats the way --outReadsUnmapped param is used. You have to say Fastx , and not an output file name. Refer to STAR manual plz

[WouterDeCoster]

Can you be more specific about the time taken by STAR? Was it surprisingly short or long? How long? How many reads is the input?

[bandanaschapagain]

I am using paired reads to be aligned to the reference genome.My reads are of 2.9 GB. It is very fast for aligning the reads and also when I check the log file, it says that there are no reads that did not match to the reference genome. for building the reference index I used ./STAR --runMode genomeGenerate --genomeFastaFiles /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/Brome_mosaic_virus/Brome_mosaic_virus_uid15052/NC_002026.fna --sjdbGTFfeatureExon /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/Brome_mosaic_virus/Brome_mosaic_virus_uid15052/NC_002026.gtf --genomeDir /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/BMV_002026_in command. Please Help me with this. Regards, Bandana

[GenoMax]

Since you are just generating the STAR index with this command there were no reads to align. Now you are ready to do the actual alignment using STAR (step 3 in STAR manual).

[bandanaschapagain]

I am doing the read alignment using step 3 but its giving me error as: segmentation fault core dump/ I am really getting confused since so long. Please anyone can help me with this.

[GenoMax]

How much memory do you have available? Since you were able to generate genome indexes your STAR install appears to be working correctly. Can you provide the command you are using for the alignment?

[bandanaschapagain]

I am using this command for the alignment.Please help with this.I have no idea how to correct it.

./STAR --genomeDir /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/Brome_mosaic_virus/Brome_mosaic_virus_INDEX --readFilesIn /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/RNA_Virus_Josef/reads/BMV/JB-BMV_S3_L001_R1_001.fastq /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/RNA_Virus_Josef/reads/BMV/JB-BMV_S3_L001_R2_001.fastq --outReadsUnmapped Fastx

[GenoMax]

That command looks ok, assuming your indexes have been created properly. Can you list output of ls -sh1 /home/yyin/RNA_Virus_Josef/RNA_Virus_Josef/Brome_mosaic_virus/Brome_mosaic_virus_INDEX

[GenoMax]

bbmap (splice aware aligner) from BBMap suite with the outu= option will capture reads that do not align.

[bandanaschapagain]

Thank you for your time and response.

[WouterDeCoster]

Please try to use a more informative title for your question, e.g. RNA-seq aligner and extracting unaligned reads.

[bandanaschapagain]

Thank you for your suggestion.I will improve my post,for sure!

Answer 1

[Ming Tommy Tang]

my github repo may help you https://github.com/crazyhottommy/RNA-seq-analysis