SEQanswers

Go Back   SEQanswers > General



Similar Threads
Thread Thread Starter Forum Replies Last Post
Conversion of .fastq to .txt RNA-seq files for EdgeR package Hamid Reza RNA Sequencing 5 11-19-2015 08:00 PM
Conversion from ‘qseq.txt’ to ‘fastq’ format joseph Bioinformatics 37 06-25-2014 11:30 PM
Conversion of qseq.txt format to fastq rakeshponnala Illumina/Solexa 7 01-08-2014 08:40 AM
seq.txt, qseq.txt and fastq NicoBxl Bioinformatics 5 01-03-2014 09:35 AM
Illumina S_sequence.txt is a fastq format? [email protected] Bioinformatics 1 04-21-2010 08:24 PM

Reply
 
Thread Tools
Old 03-30-2017, 05:52 AM   #1
iltisanni
Junior Member
 
Location: Mainz, Germany

Join Date: Mar 2017
Posts: 4
Question Illumina MiSeqFGx - fastq vs. StrResults.txt

Hey,
I'll write my Bachelor Thesis in a few weeks and I'm new to NGS, so maybe this is quite a noob question - however, these ar my problems:
1)
We are sequencing different markers like D3S1358, D22S1045 and so on with our new Illumina MiSeqFGx. Now I opened the Alignment/Report/StrResults.txt and found a result like the following for one sample:

Marker Allele Depth Sequence
D22S1045 16 23 ATTATTATTATTATTATTATTATTATTATTATTATTATTACTATTATT
D22S1045 17 78 ATTATTATTATTATTATTATTATTATTATTATTATTATTATTACTATTATT

OK, so this sample has Alleles 16,17 for D22S1045 marker.
Now I thought that all of the primer matching Sequences are stored in the .fastq files, so I started searching for the sequences in the .fastq files but havent found them.
(grep -c ATTATTATTATTATTATTATTATTATTATTATTATTATTATTACTATTATT Sample.fastq/data --> Result = 0)

What am I misunderstanding, I thought the reported sequences must be found in the .fastq file? Can anybody help?

2)
We determined the alleles with capillary electrophoresis before and now with NGS we have some other alleles as result for some samples. Sometimes Alleles determined with capillary electrophoresis arent even found by NGS and sometimes NGS finds more alleles for the same marker and the same sample than capillary electrophoresis. The StrResults.txt for example shows up 4 possible alleles and takes those with the highest depth value which is OK but someimes it only takes the one allele with the highest depth value ignoring the ones with slightly lower depth... I just dont get it...


Sorry for my bad english
iltisanni is offline   Reply With Quote
Old 03-30-2017, 09:36 PM   #2
SOLiDance
Member
 
Location: CAS

Join Date: Jun 2010
Posts: 27
Default

1. Have you tried with the reverse-complement sequence of your target?
2. It should be related with threshold setting of the UAS software suite on FGx, I highly recommend you to read its user guide through first (Universal Analysis
Software Guide). You can easily download it on illumina's website or ask for help via tech support hotline.
SOLiDance is offline   Reply With Quote
Old 03-31-2017, 12:00 AM   #3
iltisanni
Junior Member
 
Location: Mainz, Germany

Join Date: Mar 2017
Posts: 4
Default

Thx for your quick reply :-)

1. Thx. Haven't tried that so far. I'll try today.
2. Yes... that's what I suggested... I have to speak with our Guy who sets up the FGx
iltisanni is offline   Reply With Quote
Old 03-31-2017, 12:50 AM   #4
iltisanni
Junior Member
 
Location: Mainz, Germany

Join Date: Mar 2017
Posts: 4
Default

Quote:
Originally Posted by SOLiDance View Post
1. Have you tried with the reverse-complement sequence of your target?
I deleted all lines with @ and + and the quality line with sed, reversed the sequences with | rev and created the complementary sequene with sed. (so far nothing wrong?)
Now my grep finds more than the StrResults.txt tells me...
StrResults.txt says there are 23 times the sequence with depth=16 and 78 times the sequence with depth=17 but I found 114 times depth 16 and 89 tomes depth 17...

Any further ideas?
iltisanni is offline   Reply With Quote
Old 03-31-2017, 04:00 AM   #5
iltisanni
Junior Member
 
Location: Mainz, Germany

Join Date: Mar 2017
Posts: 4
Default

Another, but actually the main problem is:

The DNA-Profile of a sample clearly shows that one sample has the alleles 17/19 (capillary electrophoresis) for the marker D22S1045 but NGS only reports 17/17. NGS doesn't find the 19 allele at all...

The same for an other sample where 17/17.3 (capillary electrophoresis) are the alleles shown in the DNA-Profile for D1S1656 but only 16 and 17 is found by NGS.... Why???

Last edited by iltisanni; 03-31-2017 at 05:04 AM.
iltisanni is offline   Reply With Quote
Reply

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 08:40 PM.


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