Lower-case Nucleotides in Brucella Reads

[cscswimmer227]

Hi,
I'm very new to the community and am currently working on a new, novel assembler. I came across an interesting read in a Brucella Suis data set (sequenced with 454 Titanium FLX) and can't figure it out. In the read:

TTcCATNCGCCGGGCAGCGANGCATTGCCNTTGCCGAaNNCANNGNTNTTCNTCGGCAGGCC

What do the lower-case letters represent? Is this an issue of masking? Therefore, is there an unknown length of C's and A's in that particular read?

I appreciate any help! Thanks!

[martin2]

You should have described how you got the sequence. I suspect you work with fasta+qual or fastq data. I remember discussion whether AAAAa is represented by Roche software as 40 40 40 40 40 5 qualities or whether in contrast, it is somewhat gradually decreasing series of quality value numbers. That is probably what you are observing. But that in turn it tells me you extracted the SFF files using some non-Roche software. If I am right, at all.

[cscswimmer227]

Precisely, I am using fasta+qual files. I didn't sequence it myself though. I am using the Brucella Suis data from http://www.cbcb.umd.edu/research/benchmark.shtml.

To summarize, is the lower-case nucleotide just very low quality?

[flxlex]

If the reads sequence has come 'directly' from 454, a lower case nucleotide has a phred quality score below 40. The read you show has an unusually high number of 'N's, by the way...