SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Uniquely mapped reads and difference for single end and paired end reads gene_x Bioinformatics 2 01-13-2015 01:55 PM
Reads mapped to another chromosome in paired-end data of RNA-seq int11ap1 RNA Sequencing 1 04-21-2014 11:00 AM
How to count number of mapped paired-end and single-end rna-seq reads repinementer Bioinformatics 8 01-06-2013 06:06 AM
paired-end reads mapped to genome.. gene with only one direction of paired-end reads? danwiththeplan Bioinformatics 2 09-22-2011 03:06 AM
Paired-end reads mapped 50% while each pair reads mapped 80% zack80.liu Bioinformatics 3 03-03-2011 02:06 AM

Reply
 
Thread Tools
Old 01-05-2016, 06:41 AM   #1
Alex852013
Member
 
Location: Germany

Join Date: Jan 2013
Posts: 17
Default Problem somewhere during paired-end seq analysis -> no reads mapped

Hello everybody,

this is my fist time to try to handle to analyze paired-end sequencing files.
Finally BamCoverage complained there are no mapped reads in my Bam file. Also IGV doesn't show any reads.
I really don't know, where i lost my reads.
Here is what i did:

# Quality an adaptor trimming with trim galore:

trim_galore ../paired_1.fastq ../paired_2.fastq -q 20 --paired --phred33

# Mapping with bowtie

bowtie_opts="-p 4 -m 3 -S"
bowtie_index="path/6008.5_BAC.ebwt"
bowtie $bowtie_opts $bowtie_index -I 0 -X 700 -1 ../paired_1 .fq -2 ../paired_2.fq > paired.SAM

# Mapping report:
# reads processed: 4743277
# reads with at least one reported alignment: 1979307 (41.73%)
# reads that failed to align: 2758162 (58.15%)
# reads with alignments suppressed due to -m: 5808 (0.12%)
Reported 1979307 paired-end alignments to 1 output stream(s)

(-> 42 % of mapped reads might seem problematic. Since the sequenced plasmid was extracted from E.coli, there is a lot of contamination.)

# SAMs to BAMs

samtools view -bS ../paired.SAM > paired.BAM
samtools index paired.BAM


# BamCoverage
BamCoverage -b paired.BAM -o BamCoverage/paired.bw

This does not work and results in the following error message:

"Samtools reports that the number of mapped reads is zero for the file paired.BAM. Please check that the file is properly indexed and that it contains mapped reads."

When i google for this error message i cannot find a helpful answer. Maybe someone knows what is wrong. Since 1979307 reads could be mapped, i don't get why zero reads should be mapped.
Thanks a lot, Alex
Alex852013 is offline   Reply With Quote
Old 01-05-2016, 07:05 AM   #2
Alex852013
Member
 
Location: Germany

Join Date: Jan 2013
Posts: 17
Default

I got it on my own, after a short break. I forgot to sort the bam files...
Alex852013 is offline   Reply With Quote
Old 01-06-2016, 02:41 AM   #3
colindaven
Senior Member
 
Location: Germany

Join Date: Oct 2008
Posts: 392
Default

One point - it looks like you are using bowtie for alignment, which is obsolete for most purposes. Bowtie2 is a much more accurate aligner, and can detect indels. BWA is another great alternative, as is (commercial) Novoalign.
colindaven is offline   Reply With Quote
Old 01-07-2016, 05:40 AM   #4
Alex852013
Member
 
Location: Germany

Join Date: Jan 2013
Posts: 17
Default

Thanks for the hint. My adviser told me it is sufficient to use Bowtie 1.
I tested Bowtie 2 now and i got 10 % more alligned reads. For another sample even 23 %, which is really a lot!
Thanks again!
Alex852013 is offline   Reply With Quote
Old 01-07-2016, 08:25 AM   #5
westerman
Rick Westerman
 
Location: Purdue University, Indiana, USA

Join Date: Jun 2008
Posts: 1,104
Default

Let's not forget BBMap as a good non-commercial alternative.
westerman is offline   Reply With Quote
Old 01-07-2016, 10:07 AM   #6
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,579
Default

@Alex852013: A large % of reads is still failing to align (if you are only getting 23% of reads to align) so you may want to investigate why that is so. Quick blasting @NCBI should give a clue as to whether you data has unexpected contamination.
GenoMax is offline   Reply With Quote
Old 01-07-2016, 10:12 AM   #7
westerman
Rick Westerman
 
Location: Purdue University, Indiana, USA

Join Date: Jun 2008
Posts: 1,104
Default

Quote:
Originally Posted by GenoMax View Post
@Alex852013: A large % of reads is still failing to align (if you are only getting 23% of reads to align) so you may want to investigate why that is so. Quick blasting @NCBI should give a clue as to whether you data has unexpected contamination.
Didn't he say 23% more not 23% total? In any case the original post talked about mapping a mixture of plasmid and E.coli reads against the plasmid and thus he expected a low percentage.

Last edited by westerman; 01-07-2016 at 10:13 AM. Reason: Added question mark.
westerman is offline   Reply With Quote
Old 01-07-2016, 10:25 AM   #8
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,579
Default

I missed that fact from the original post. @Rick: Thanks for pointing that out. If true #6 can be safely ignored.
GenoMax is offline   Reply With Quote
Old 01-08-2016, 12:53 AM   #9
Alex852013
Member
 
Location: Germany

Join Date: Jan 2013
Posts: 17
Default

Yes, it meant 23 % more, which makes 96 % in total. Perfect!
Nevertheless, thanks for caring!
Alex852013 is offline   Reply With Quote
Reply

Tags
paired-end, sequencing, zero reads

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 05:48 AM.


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