SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Number of Reads hamcan Bioinformatics 9 12-20-2016 04:20 PM
Number of reads anj_1 Illumina/Solexa 3 11-06-2014 03:58 AM
Tophat 2.0.0 number of reads EGrassi Bioinformatics 8 11-09-2012 12:34 AM
Number of Reads Rfriedman Bioinformatics 0 01-11-2012 02:43 PM
number of reads m_elena_bioinfo Bioinformatics 2 07-20-2010 09:47 AM

Reply
 
Thread Tools
Old 01-18-2017, 11:11 AM   #1
hamcan
Member
 
Location: Toronto

Join Date: Nov 2016
Posts: 19
Default Number of Reads Increasing

Hi there,
I trimmed my reads and then merged them using FLASh. However, I found that the number of reads before processing (trim and merge) is lower than the number of reads after processing.
Is there an explanation for this?
Thanks
hamcan is offline   Reply With Quote
Old 01-18-2017, 11:23 AM   #2
Brian Bushnell
Super Moderator
 
Location: Walnut Creek, CA

Join Date: Jan 2014
Posts: 2,695
Default

When you merge a read pair, you turn 2 reads into 1 read. Therefore, you should expect the number of reads to decrease.
Brian Bushnell is offline   Reply With Quote
Old 01-18-2017, 02:10 PM   #3
hamcan
Member
 
Location: Toronto

Join Date: Nov 2016
Posts: 19
Default

i know i should expect a decrease but i'm getting an increase. do you know why?
hamcan is offline   Reply With Quote
Old 01-18-2017, 02:34 PM   #4
Brian Bushnell
Super Moderator
 
Location: Walnut Creek, CA

Join Date: Jan 2014
Posts: 2,695
Default

Perhaps you can post your exact commands used at each step, as well as how you are calculating the number of reads?
Brian Bushnell is offline   Reply With Quote
Old 01-19-2017, 10:25 AM   #5
hamcan
Member
 
Location: Toronto

Join Date: Nov 2016
Posts: 19
Default

First step I'm doing is using Trimmomatic to trim and remove my adapters using the PE setting:

java -jar trimmomatic-0.36.jar PE File1_L001_R1_001.fastq File1_L001_R2_001.fastq /home/rachelle/File1_R1paired.fastq File1_R1unpaired.fastq File1_R2paired.fastq File1_R2unpaired.fastq ILLUMINACLIP:/usr/local/trimmomatic/adapters/NexteraPE-PE.fa:2:30:10:6 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:25 MINLEN:36

Afterwards, I take R1paired and R2 paired and merge them using FLASh with a minum of overlap of 15.
I then concatenate the out.extended frags with the two notcombined files for a final file that is then concatenated with R1UNpaired and R2UNpaired.

That final file has more reads than the beginning.
I looked into it some more and saw that my reads are increasing after FLASh. I am assuming it has to do with concatenation of the two notCombined files. Does anyone know what those two files are? And if I should be concatenating them with my out.extendedfrags file?

Thanks
hamcan is offline   Reply With Quote
Old 01-19-2017, 10:33 AM   #6
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,574
Default

Best option would be to merge first (use bbmerge.sh from BBMap) and then trim (bbduk.sh from BBMap).

The only way number of reads is going to increase is if you double dip into the read pool. You must be somehow doing that. Since your merged reads become "single-end" you should not merge them with remaining PE reads.

Last edited by GenoMax; 01-19-2017 at 10:43 AM.
GenoMax is offline   Reply With Quote
Old 01-19-2017, 12:14 PM   #7
HESmith
Senior Member
 
Location: Washington DC

Join Date: Oct 2009
Posts: 490
Default

Paired-end reads are typically counted once (unless it's the Illumina marketing team). Merged reads will also be counted once after FLASH, but unmerged reads now become two single-end reads and are counted twice.
HESmith is offline   Reply With Quote
Old 01-19-2017, 12:27 PM   #8
hamcan
Member
 
Location: Toronto

Join Date: Nov 2016
Posts: 19
Default

that makes perfect sense!!! Thank you so much
hamcan is offline   Reply With Quote
Reply

Tags
bioinformatics, dna, merge, sequencing, trim

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 01:02 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO