I am new to the NGS analysis. I need help to solve this problem. As shown in my previous emial/question shown below, I have some paired-end datasets at FASTQ format, and I have problem to split each of these datasets into two datasets (one forward and one reverse). Jennifer instructed me to assign the datatype to be fastqsanger first and then run 'Manipulate FASTQ'. I have two questions: 1) Now that the datasets were already split into forward and reverse reads when extracted in FASTQ format from the SRA, can I use them just as single end data? 2) If I do need to split each dataset into two datasets, how should I choose the settings when I run "Manipulte FASTQ"? Thanks. Jianguang ///////////////////////// On 8/10/12 7:21 AM, Du, Jianguang wrote:

I have problem to split a paired-end FASTQ dataset into two separate datasets. In order to explain the problem clearly, I list the detail of what I did with my dataset:

Step 1) My aim is to compare datasets for the differential alternative splicing. I downloaded paired-end datasets at FASTQ format from SRA of NCBI as original data.

Below is part of my paired-end FASTQ dataset that I downloaed from SRA of NCBI, Does this dataset look OK?

@SRR192532.1.1 HWI-EAS269:1:4:655:110.1 length=35 GTTTTCTGAGTGAGAAAAGGTGTGTTTGGAGTTTG +SRR192532.1.1 HWI-EAS269:1:4:655:110.1 length=35 I28II;II*2/<5:++,(..*943F@I.('+.35' @SRR192532.1.2 HWI-EAS269:1:4:655:110.2 length=35 AAAGATGTTAGTGTTTTATACGGAAAGGATATCTC +SRR192532.1.2 HWI-EAS269:1:4:655:110.2 length=35 9+*9+7@?F1206,IGI+D122&/0++-.>+6/@?

Step 2) Then I performed FASTQ groomer at setting as follows:

a) Input FASTQ quality scores type: Illumina 1.3-1.7

b)Advanced Options: Hide Advanced Options.

Did I choose the right setting for FASTQ groomer? Should I use Advanced Options? If yes, what is the setting for Advances Options?

Below is part of groomed dataset:

@SRR192532.1.1 HWI-EAS269:1:4:655:110.1 length=35 GTTTTCTGAGTGAGAAAAGGTGTGTTTGGAGTTTG +SRR192532.1.1 HWI-EAS269:1:4:655:110.1 length=35 *!!**!**!!!!!!!!!!!!!!!!'!*!!!!!!!! @SRR192532.1.2 HWI-EAS269:1:4:655:110.2 length=35 AAAGATGTTAGTGTTTTATACGGAAAGGATATCTC +SRR192532.1.2 HWI-EAS269:1:4:655:110.2 length=35 !!!!!!!!'!!!!!*(*!%!!!!!!!!!!!!!!!!

Does the groomed data look right? Is number represnting the member of a pair correct? Here they are ".1" and ".2", should they be "/1" and "/2"?

Step 3) Then I ran FASTQ splitter with the groomed files. There is not setting for the splitter. I chose the right groomed file and then click "Excute". Below is the description of the splitted dataset:

empty format:fastqsanger, database:hg19 Info: Split 0 of 15277248 reads (0.00%).

Please help me dela with this problem.

Thanks.

Jianguang Du