How To Evaluate 454 Sequences

Question

[Sashi Kiran Challa]

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

[Aaronquinlan]

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

Answer 1

[Science_Robot]

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.

Answer 2

[Casbon]

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.

Answer 3

[Val]

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, ...

Answer 4

[Navi]

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