ALLPATHS-LG: Using different quality scorings

[jpummil]

Working on creating my .csv files for a large assembly in ALLPATHS and I recall someone telling me that ALLPATHS needs special attention when utilizing libraries with different phred scorings? How do I incorporate that into my input files?

Illumina 1.5 = phred 64?
Illumina 1.9 = phred 33?

Currently using a .ppt by Mike Schatz as a reference as this is my first use of ALLPATHS and it is a bit different than other assemblers I have used in the past.

[jpummil]

Wow! 140+ views thus far...my question must be pretty "off the beaten path".

I found a reference to this on the Trinity mailing list of all places:

http://sourceforge.net/p/trinityrnaseq/mailman/message/28066240/

Excerpt:
The PHRED+33 vs +64 issue is known. Sanger uses +33, and Illumina uses +64
(1.3+, 1.5+). However, Illumina 1.8+ uses phred+33 again
(http://en.wikipedia.org/wiki/Fastq). Since that's the default in our
environment here, it has become the default for FastqToFastbQualb. In most
situations one can auto-detect which is which, but not in general. Therefore
the user must specify PHRED_64=True if that is the case.

[jpummil]

Looks like best bet here is to simply convert the old Illumina 1.5 Phred 64 scored files to the newer Illumina 1.9 Phred 33 scoring.

LOOKS...like bbmap will do this, but I'll have to try it in a bit.

Maybe: bbmap qin=64 old-phred.fastq qout=33 phred-new.fastq

[jpummil]

So, I ended up trying seqtk as a colleague had suggested that first...

seqtk seq -VQ64 s1_R1_PE.fastq > s1_R1_PE_Phred33.fastq

A quick check of the resulting file with FastQC seems to confirm the change:

##FastQC 0.11.1
>>Basic Statistics pass
#Measure Value
Filename s1_R1_PE_Phred33.fastq
File type Conventional base calls
Encoding Sanger / Illumina 1.9
Total Sequences 98578990
Sequences flagged as poor quality 0
Sequence length 101
%GC 38
>>END_MODULE

However, Phred64 must contail a LOT more text as the file sizes diminished significantly as a result of the conversion:

-rw-r--r-- 1 jpummil jpummil 27G Mar 25 2014 s1_R1_PE.fastq
-rw-rw-r-- 1 jpummil jpummil 23G Oct 8 14:12 s1_R1_PE_Phred33.fastq