Where is the read?

[drgoettel]

Hello,

I have "lost" one read in a 454 assembly. That read is in the .sff used to run the assembly.
It's also at 454ReadStatus.txt file telling that it became a Singleton

nameoftheread Singleton

It also has been trimmed:
Accno Trimpoints Used Used Trimmed Length Orig Trimpoints Orig Trimmed Length Raw Length
nameoftheread 5-270 266 5-270 266 270

I have the "allContigThresh" parameter to 100.

So anybody knows why isn't this read at 454AllContigs.fna output??
Where has it gone?

Thanks in advance.






PD: I write down the qualities extracted from the sff file, in case they can help:

Bases: tcagTCGCAAGTGCCACGACCAGAAAGAATTGATGGTGGCGGTTGTTTGCCACACGACCTCTCCCGAAGACTTTGGAGGTAATGCCGACATTGGATCTGGCAAGAACAAAGCTTAACCCTAATTTATTTTATAGCAATAGCAAGGGTTAAGCTTTGTTCTTTCCATATTTGATGTCAGCATTCCCTCCTAAGACTCAGGAATGGATGTGGAAATTGTGCTTCGCAAGAACCTACAGTCGTCGCTTCGGTATGGACAAAGCTTGAAGGTTG
Quality Scores: 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 33 20 20 23 23 22 22 31 31 33 33 35 35 35 35 35 35 36 35 35 35 35 35 35 35 35 35 31 31 31 33 35 35 35 35 33 33 33 35 35 31 31 31 33 33 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 33 33 33 35 35 35 35 35 35 35 35 35 35 35 35 33 31 31 31 33 35 35 35 35 31 31 31 35 35 31 31 31 31 17 17 17 25 24 24 25 25 25 31 20 20 20 20 35 35 35 35 33 31 31 32 35 35 35 30 30 21 21 21 21 21 33 33 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 33 33 33 35 35 35 35 35 35 35 35 35 35 35 35 33 33 33 35 35 35 35 35 35 35 35 35 35 35 35 35 35 33 33 33 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 33 31 31 33 34 34 34 34 34 34 27 27 27 33 34 34 34 34 34 34 34 34 34 34 31 31 31 26 26 26 28 28 28 28 29 28 28 28 28 28 28 28 28 28 22 22 22 22 22 22 22

[kmcarr]

The answer is there in the 454ReadStatus.txt file. This read is a "Singleton" meaning it was not assembled into any contig, that's why it's not in the 454AllContigs.fna. That file does not include singletons.

If your assembly is typical you should have many more than just one Singleton remaining. Look at the readStatus section of your 454NewblerMetrics.txt file to see the total of Assembled, Singleton, et al. in your assembly.

If you want to recover a file of the unassembled (Singleton) reads you could create a text file listing their IDs (taken from the ReadStatus file) and use this list in conjunction with the sfffile and sffinfo tools to extract the singleton reads from the sff file.

[drgoettel]

numreads=1?

Thankyou for your answer
But inside 454AllContigs.fna are a lot of "contigs" with numreads=1
They are singletons to me. Aren't they??

[kmcarr]

Thankyou for your answer
But inside 454AllContigs.fna are a lot of "contigs" with numreads=1
They are singletons to me. Aren't they??

No, those are not classified as singletons. The read which makes up a single read contig will be listed as Assembled in the 454ReadStatus file.

Now you may ask, how come some reads are singletons and other assembled as single read contigs? Not knowing the internals of the gsAssembler I can't answer definitively but my guess is that these single read contigs may have been been part of larger contigs originally but these reads were ripped out during latter stages of the assembly for whatever reason. They then become classified as single read contigs vs singletons which are reads which were never included in any contig. Again, this is only a guess.