Go Back   SEQanswers > Bioinformatics > Bioinformatics

Similar Threads
Thread Thread Starter Forum Replies Last Post
Good way to align very, very short reads? bloosnail Bioinformatics 13 02-28-2017 04:53 PM
align short query against 300M Illumina reads hohllp Bioinformatics 5 11-21-2011 12:40 PM
How to map short reads to a distant genome? ynwh Illumina/Solexa 5 08-03-2011 05:56 AM
Looking for assembler to align very short reads andylai Bioinformatics 0 06-02-2011 08:58 PM

Thread Tools
Old 04-23-2018, 02:28 AM   #1
Junior Member
Location: France

Join Date: Feb 2013
Posts: 6
Default Best strategy/tools to align poor quality reads on distant/degenerate short reference

Hello everybody,

Here is my problem:
I have +/-50 samples that I sequenced to examine SNP at key positions. I know my key positions.
I have 4 reference sequences (+/-2000bp). Two of them are encoded with IUPAC nucleotide code and 2 of them with "ATGC" code. These references could be relativly far tha the obtained reads
I have high size (1000bp) amplicon paired reads data. As the amplicon size is far larger than my reads, I will work on my reads files separatly.
My reads quality is really poor, the base quality dropping rapidly below 20 around 90 bp (on 250bp reads).

What I did:
I started to aligne my reads direcly with bwa mem on my references, changeing the seeds quality and mismatch scores...
The I wanted to perform snp calling with freebayes. Unfortunatly, I don't think this is a good idea (I don't know the ploidy of my samples).
Then, I discoverd that my reads had a really poor quality, then I decided to had a first step of read cleaning, with trimmomatic. (SE -threads 8 -phred33 -trimlog trim_${readsname}.log ${file} ${OUTDATADIRECTORY}/${readsname}_001.trimmed.fastq.gz \ILLUMINACLIP:./adapters/NexteraPE-PE.fa:2:30:10 LEADING:10 TRAILING:10 SLIDINGWINDOW:4:15 AVGQUAL:30 MINLEN:36)
After talking with some friends, and seeing the reads siez dropping, I decided to use bwa aln and perform the snp calling with sammtools/bcftools. But the alignement parameters are harder to correctly set.

Then, my questions:
- Are my pipeline steps good? must I clean my reads before align them, or the alignement will take quality into account andthis step is useless?
- Which programs must I used for this different steps? I saw some peaople use Mosaik, ssaha, bfast, novoalign, etc... Which one is the best for my particular problem?
- Which snp calling method/program must I used? as I am focusing on specific known position, is a Bayesian haplotype-based usefull or mpilup is enough?
- Do you have any advice for me? (this is my first experiment with this kind of data, before I worked on high quality human data...).

As said a famous people "Help me, Obi-Wan Kenobi. You're my only hope."

Thx in advance.

Last edited by denisDDS; 04-23-2018 at 03:31 AM.
denisDDS is offline   Reply With Quote

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 03:39 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO