In Cufflinks, when using the -g or -G for the GTF guide, is there a difference in using upper or lower case? (There are two separate entries in the Manual) ... If there is a difference, how could this impact the discovery of novel Isoforms?
with the option -G, cufflinks will not look for any novel transcript. only the reads that can be mapped to the GTF intervals will be retained. with option -g, cufflinkds will look for novel transcripts and it will give an output of both the reads mapped to the GTF file and also the reads mapped somewhere else in the genome, that form a likely transcript
-G option won't look for novel isoforms. It will simply ignore alignments that are not present in your annotation file. On the other hand -g option will get you the expression estimates for novel isoforms too.
From Cufflinks manual:
-G/--GTF <reference_annotation.(gtf gff)=""> Tells Cufflinks to use the supplied reference annotation (a GFF file) to estimate isoform expression. It will not assemble novel transcripts, and the program will ignore alignments not structurally compatible with any reference transcript.
-g/--GTF-guide <reference_annotation.(gtf gff)=""> Tells Cufflinks to use the supplied reference annotation (GFF) to guide RABT assembly. Reference transcripts will be tiled with faux-reads to provide additional information in assembly. Output will include all reference transcripts as well as any novel genes and isoforms that are assembled.