paired end data not aligned in NextSeq 500

Julia_m · 06-28-2016, 03:07 AM

Hello!!
I'm quite new in NGS and I got these fastq files (read1 and read2) from illumina nextseq 500.
Now the problem is that in somehow the two file are not aligned, I mean, I checked for the coordinates in read1 and read2 on the same row and it appears that sometimes are not the same!
Since I'm quite new, this can be possible? or anyway is there any tools or bash script that can help me in the alignment of these files?

Hope seriously in somebody that can help me!

Krish_143 · 06-28-2016, 03:23 AM

Hi Julia_m,

Is that Genomic data or RNAseq - transcriptomic data. Okay !

If it was not aligning that could mean.. paired read was not found in one of the other file.

Probably you might have the trimmed data and considered all the ones. If it was trimmed you should keep the read that present in both the files. (Check any trimming software eg: cutadapt and try mapping)

Hope it works.

Julia_m · 06-28-2016, 03:51 AM

cutadapt is for adaptor cutting isn't it?
i mean my problem is quite different, I have two fastq files like these:
READ1.fastq
@NB501365:8:HF3HLAFXX:1:11101:22082:1033 1:N:0:CGAGTA
READ2.fastq
@NB501365:8:HF3HLAFXX:1:11101:22082:1033 2:N:0:CGAGTA

the coordinates 22082:1033 for read1 and 22082:1033 for read2 shouldn't be the same in paired ends?

Julia_m · 06-28-2016, 03:56 AM

sorry big mistake READ2.fast has the following header:
@NB501365:8:HF3HLAFXX:1:11101:64563:1033 2:N:0:CGAGTA

dpryan · 06-28-2016, 04:11 AM

Your data looks as it should. The "1:N:0:CGAGTA" portion, as an example, just says "I'm read 1, I didn't fail quality control filtering on the machine, and my barcode was CGAGTA". Read 2 should look the same (and have the same read name), with the exception that there's a 2 rather than a 1 in the second block of text.

So go ahead and quality/adapter trim this dataset (e.g., with "Trim Galore!" or trimmomatic) and then use an aligner (bowtie2, bbmap, hisat2, bwa, STAR, etc.).

Edit: Oh, if the read names are really different (I just now noticed your most recent post) then you'll need to resync the files. There's a tool in BBMap that can do this for you (it has a LOT of convenient tools).

GenoMax · 06-28-2016, 04:34 AM

As @Devon said your R1/R2 files may be out of sync. You can use repair.sh from BBMap suite to re-sync the files.

You will find example of that command line and lots of other things BBMap suite can do in this thread.

sklages · 07-12-2016, 11:29 PM

It would still be interesting, what has caused the reads to be out-of-sync.
Have these reads been pre-processed with some tool before or is this the data that has been sent by sequencing provider?

Fixing is important, avoiding, or knowing how to avoid it, is more important ;-)

Just my 2p.

Julia_m · 07-23-2016, 01:44 AM

problem solved!! seems that something happened during the unzipping process (I don't know why), if you process everything directly from the gzip file it's fine!.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
A first look at Illumina’s new NextSeq 500	AllSeq	Vendor Forum	111	03-12-2020 03:25 AM
Weird FastQC reports on NextSeq 500 data	nucleolus	Illumina/Solexa	4	11-05-2015 03:13 AM
A server for processing data from Nextseq 500？	johnsunx1	Core Facilities	5	08-31-2015 04:52 AM
100 Gb Data/Day – Nextseq 500 Sequencing Services Now Available on Genohub	Genohub	Vendor Forum	3	04-24-2014 09:28 AM
Paired-end Bam from single-end aligned sam	ramouz87	Bioinformatics	4	08-17-2011 01:55 PM

06-28-2016, 03:07 AM	#1
Julia_m Junior Member Location: stockholm Join Date: Jun 2016 Posts: 4	paired end data not aligned in NextSeq 500 Hello!! I'm quite new in NGS and I got these fastq files (read1 and read2) from illumina nextseq 500. Now the problem is that in somehow the two file are not aligned, I mean, I checked for the coordinates in read1 and read2 on the same row and it appears that sometimes are not the same! Since I'm quite new, this can be possible? or anyway is there any tools or bash script that can help me in the alignment of these files? Hope seriously in somebody that can help me!

06-28-2016, 03:23 AM	#2
Krish_143 Member Location: Sweden Join Date: Jan 2012 Posts: 45	Hi Julia_m, Is that Genomic data or RNAseq - transcriptomic data. Okay ! If it was not aligning that could mean.. paired read was not found in one of the other file. Probably you might have the trimmed data and considered all the ones. If it was trimmed you should keep the read that present in both the files. (Check any trimming software eg: cutadapt and try mapping) Hope it works. __________________ Krishna

06-28-2016, 03:51 AM	#3
Julia_m Junior Member Location: stockholm Join Date: Jun 2016 Posts: 4	cutadapt is for adaptor cutting isn't it? i mean my problem is quite different, I have two fastq files like these: READ1.fastq @NB501365:8:HF3HLAFXX:1:11101:22082:1033 1:N:0:CGAGTA READ2.fastq @NB501365:8:HF3HLAFXX:1:11101:22082:1033 2:N:0:CGAGTA the coordinates 22082:1033 for read1 and 22082:1033 for read2 shouldn't be the same in paired ends?

06-28-2016, 03:56 AM	#4
Julia_m Junior Member Location: stockholm Join Date: Jun 2016 Posts: 4	sorry big mistake READ2.fast has the following header: @NB501365:8:HF3HLAFXX:1:11101:64563:1033 2:N:0:CGAGTA

06-28-2016, 04:11 AM	#5
dpryan Devon Ryan Location: Freiburg, Germany Join Date: Jul 2011 Posts: 3,480	Your data looks as it should. The "1:N:0:CGAGTA" portion, as an example, just says "I'm read 1, I didn't fail quality control filtering on the machine, and my barcode was CGAGTA". Read 2 should look the same (and have the same read name), with the exception that there's a 2 rather than a 1 in the second block of text. So go ahead and quality/adapter trim this dataset (e.g., with "Trim Galore!" or trimmomatic) and then use an aligner (bowtie2, bbmap, hisat2, bwa, STAR, etc.). Edit: Oh, if the read names are really different (I just now noticed your most recent post) then you'll need to resync the files. There's a tool in BBMap that can do this for you (it has a LOT of convenient tools).

06-28-2016, 04:34 AM	#6
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,089	As @Devon said your R1/R2 files may be out of sync. You can use repair.sh from BBMap suite to re-sync the files. You will find example of that command line and lots of other things BBMap suite can do in this thread.

07-12-2016, 11:29 PM	#7
sklages Senior Member Location: Berlin, DE Join Date: May 2008 Posts: 628	It would still be interesting, what has caused the reads to be out-of-sync. Have these reads been pre-processed with some tool before or is this the data that has been sent by sequencing provider? Fixing is important, avoiding, or knowing how to avoid it, is more important ;-) Just my 2p.

07-23-2016, 01:44 AM	#8
Julia_m Junior Member Location: stockholm Join Date: Jun 2016 Posts: 4	problem solved!! seems that something happened during the unzipping process (I don't know why), if you process everything directly from the gzip file it's fine!.