4
$\begingroup$

I need to have a RNA-Seq dataset and therefore, I've visited the following site NCBI-geo C. Elegans

In the Supplemantary file part, I clicked the SRP/SRP051/SRP051702 ftp and downloaded sra file. Then I need to convert it to fastq file format. For this purpose I've heard that there is a sra-toolkit and within it there is fastq-dump exacutable. However in order to use it, first of all I have to figure out whether my sra file contains paired-end or single-end read data. So my question is that how could I know the type of reads in the dataset (in the first link) ?

Without any information, I used --split_files flag of fastq-dump exacutable and it generated two 14 Gb (both of them are exactly 14.346.367.840 bytes) files and their name are SRR1741330_1.fastq and SRR1741330_2.fastq Does it mean my dataset is paired end ?

As an another question (different but related with part 1). In the SRR1741330_1.fastq file, at some lines, sequences contains different characters such as CCCFFFFFGFHHHGJJJJI#1?FEIGGI... Before looking into these files, I have thought that these sequence lines should only contain the letters of A,G,T and C . What are these F,H,J,I,#,? etc..

My questions might be trivial and unmeaningful but since I'm a totally new person in this area, I could not understand them.

EDIT

New question

I wonder one more thing, since its related with previous part of my question I asked it here instead of creating new question. After I generate fastq file with dump_fastq executable, all the sequences are length of 50. Does it because of parameters of the dump_fastq executable or its related with original sra file? In other words, could I increase this length?

Improve this question
$\endgroup$

1 Answer 1

Reset to default
2
$\begingroup$

To find out whether a dataset was paired-end or single-end, go to SRA, click on a run, and look under "Library". Paired-end datasets will typically have "Layout: paired". Note that people don't always mark this correctly, which causes no end of headaches.

Regarding lines like "CCCFFFFFGFHHHGJJJJI#1?FEIGGI", that's the quality score line. Look at the fastq article in wikipedia for more details.

Edit: Regarding the updated portion of your question, no you can not increase this length. The sequences produced by Illumina machines are a fixed length and that length (or whatever length was submitted) is what you're getting. As a general rule, you'll get whatever sequence was updated (unless you forget the --split3 (or whatever it's called) option).

Improve this answer
$\endgroup$
7
  • $\begingroup$ @Devon_Ryan thanks for your help, by mean go to SRA, I think you refer to sra file that I downloaded from the link I provide above,right? $\endgroup$
    – stackunderflow
    May 8, 2015 at 17:16
  • $\begingroup$ Okey, now I found it, it is in the browser $\endgroup$
    – stackunderflow
    May 8, 2015 at 17:33
  • 1
    $\begingroup$ Exactly, I mean the webpage for a run on the SRA website. The same information is typically available in GEO too (click on one of the samples and read the "Data Processing" section), but it's typically a bit faster to just go to SRA. $\endgroup$
    – Devon Ryan
    May 8, 2015 at 17:54
  • $\begingroup$ @Devon_Ryan thanks again, could you look at my question again, I've updated it $\endgroup$
    – stackunderflow
    May 8, 2015 at 17:57
  • 1
    $\begingroup$ I've updated my reply. The short answer is that you're getting what was uploaded, which is typically all that was sequenced. $\endgroup$
    – Devon Ryan
    May 8, 2015 at 18:30

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.