I want to know about the actual function of Bowtie?

Question

[PK]

Hi friends, I am learning RNA_Seq. I want to know the actual function of bowtie. For my understanding bowtie takes reference genome file (.fa) as an input after that it generates .bt2 files as an output. In my point of view this step for indexing (string compression for aligning). Bowtie also used for aligning at this point I was confused. In what step bowtie do align and what are the input files. please explain. I know this is a simple question. But I got stuck.

[Brian Bushnell]

Please don't tag your question as "software error" if there is no software error. I have corrected your tags.

Answer 1

[JC]

Bowtie is a set of programs, "bowtie-build" creates the index from a reference genome. After having your index, "bowtie" aligns short sequences (in Fasta or Fastq format) into the index.

[PK]

1. Thanks for your answer. bowtie-build creates indexing so that file stored in .bt format. I am using this command (/opt/bowtie2_2_2_1/bowtie2-build ~/ACTREC_RNA/Ref/genome.fa ~/ACTREC_RNA/bwt/genome/genome). My doubt is without fastq file how to do bowtie align.

before running tophat i import bowtie. After importing bowtie i run Tophat

/opt/tophat_2_0_11/tophat -p 16 -r 60 --library-type fr-firststrand -o TopHat_OUPUT -G ~/ACTREC_RNA/Annotation/genes.gtf ~/ACTREC_RNA/bwt/genome/genome ~/ACTREC_RNA/RAW_DATA/10_P9_7_P2_GTGAAA_L005_R1_001.fastq ~/ACTREC_RNA/RAW_DATA/10_P9_7_P2_GTGAAA_L005_R2_001.fastq

I don't know what i did. So please do explain me what actually i did.

[GenoMax]

Yikes! You are using a very old version of TopHat (2+ years). So the first thing to try would be to upgrade that. Your bowtie2 is also not the latest so upgrade it while you are at it.

TopHat will take care of the actual bowtie2 alignments as long as you have bowtie2 available and properly installed.

My doubt is without fastq file how to do bowtie align.

Not sure what you are asking there. Can you clarify?

[PK]

Thank you for your reply. First point is i have to replicate the data that is why i am using tophat. I am confused at this step. In bowtie command, i didn't use any fastq file so that (My doubt is without fastq file how to do bowtie align). At which step bowtie do align. Next doubt is what is the function of Tophat.

[GenoMax]

Take a look at figure 1 in the Nature methods protocol that describes the TopHat suite in detail.

Index creation is an essential step in the preparation of reference genomes for NGS alignment. It needs to be done only once (bowtie2-build process). Once the index files are ready they are re-used for all subsequent alignments that use the bowtie2 (or TopHat2 which internally uses bowtie2 for alignments) program.

[PK]

Thank you very much. I uesd this command. In tophat it has two things tophat and tophat2. Is there any difference between tophat and tophat2. But i did tophat

/opt/tophat_2_0_11/tophat -p 16 -r 60 --library-type fr-firststrand -o TopHat_OUPUT -G ~/ACTREC_RNA/Annotation/genes.gtf ~/ACTREC_RNA/bwt/genome/genome ~/ACTREC_RNA/RAW_DATA/10_P9_7_P2_GTGAAA_L005_R1_001.fastq ~/ACTREC_RNA/RAW_DATA/10_P9_7_P2_GTGAAA_L005_R2_001.fastq