Question: How To Evaluate 454 Sequences
4
gravatar for Sashi Kiran Challa
8.7 years ago by
Portland, Oregon
Sashi Kiran Challa300 wrote:

Hi All,

I have not done a lot of sequencing or taken a look at reads obtained from 454 sequencer. I am interested in learning different methods used to evaluate the quality of the reads obtained (before assembly step), for instance removing repeats if any. Could some one please elaborate or point me to any information available on-line, on what is usually done to check the quality of reads obtained from Roche 454.

Thanks Sashi

read quality • 2.3k views
ADD COMMENTlink written 8.7 years ago by Sashi Kiran Challa300

Do you have a high-quality reference genome for your organism of interest?

ADD REPLYlink written 8.7 years ago by Aaronquinlan10k
5
gravatar for Science_Robot
8.7 years ago by
Science_Robot1.1k
Gainesville, FL
Science_Robot1.1k wrote:

You can look at the quality scores. Wikipedia actually has a great article on them.

I don't know if you have any programming experience but it's not difficult to trim/filter based on phred scores.

ADD COMMENTlink modified 6 months ago by RamRS20k • written 8.7 years ago by Science_Robot1.1k
5
gravatar for Casbon
8.7 years ago by
Casbon3.2k
Casbon3.2k wrote:

Check the filter statistics first:

  • How many reads did you get?
  • How many were lost to the different filters?
  • Did you see a high lost to mixed reads, or whatever?

Now you can look at the length of your reads. Did the trimback filter take a lot out? Are there an excess of short reads from primers, etc?

If you really want to go into detail load up the images into the run browser and check the plate layout. Sometimes you'll see bubbles or low quality regions.

Really though, to state anything else about quality needs a mapping. Then you can check error rates, etc.

ADD COMMENTlink modified 6 months ago by RamRS20k • written 8.7 years ago by Casbon3.2k
4
gravatar for Val
8.7 years ago by
Val50
Jouy-en-Josas, France
Val50 wrote:

I use FastQC to do basic QC checks. It takes quality files (fastq) as input and gives a number of statistics on your run : quality distribution, GC %, over-representated sequences, ...

ADD COMMENTlink modified 6 months ago by RamRS20k • written 8.7 years ago by Val50
1
gravatar for Navi
8.6 years ago by
Navi10
Navi10 wrote:

You can also try clustering reads using 'uclust' or 'clustalw' to try to reduce the number of total reads.

ADD COMMENTlink written 8.6 years ago by Navi10
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