Question about evaluation of 454 sequence

[lischka]

Hi all,

Lately our lab 454 sequenced a 5Mb human genome captured by nimblegen Seqcap array. And we got the sequence back 2 days ago from the sequence service. I just quickly looked at the report: the average length is a little more than 200bp and the short read % is about 50%. My question is that are these parameters within the normal range? How do you determine whether a sequence is good or bad for 454? Is there any document that I can refer to?

Thanks!

[vinay052003]

Hi,
You've got 5MB of sequence data for human genome which reflects the low coverage over the entire genome. But as you did targeted sequening (Nimblegen SeqCap array), low amount of data does make sense.
How short your reads are in "short read" category?

Anyways.. if you want to check the quality of your sequences, you can plot the average quality scores against the %length of the sequencing read. Usually Q20 reflects 99% accuracy in 454 sequencing.
One more way of quality check is to align these sequence to their targets and then plot the %identity with the number of sequences falling the in bins of %identity. Most of the sequences should show very high identity (>95%) if data is good and contains very little sequencing errors.