Catfish pooled BAC clone sequencing
A Pilot Study for Channel Catfish Whole Genome Sequencing and de novo Assembly
Twenty-four BAC clones from a large contig of catfish physic map were selected for sequencing and assembly. The BAC DNA was extracted and pooled for sequencing using one lane for Illumina sequncing and 1/4 plate for Roche 454 sequencing.
As a pilot project for whole genome sequencing of the catfish genome, here we attempt to determine the proper sequence coverage, the proper software for assembly, and various parameters used for the assembly of a BAC physical map contig spanning approximately a million of base pairs. A combination of low sequence coverage of 454 and Illumina sequencing appeared to provide the most cost-effective assembly.  Using 454 sequencing alone, a sequencing depth of 18 X was sufficient to obtain the effective assembly, whereas a 70 X Illumina appeared to provide effective sequence assembly.  Additional sequencing coverage after 18 X of 454 or after 70 X of Illumina sequencing was no longer cost effective.  A 2 X 454 sequencing, when coupled to 70 X Illumina sequencing, provided the cost-effective, good quality assembly.  With several software tested, Newbler with a seed length of 16 and ABySS with a K-value of 60 appear to be appropriate for the assembly of 454 reads alone and Illumina paired end (PE) reads alone, respectively.  Using both 454 and Illumina PE reads, a hybrid assembly strategy using Newbler for initial 454 sequence assembly, Velvet for initial Illumina sequence assembly, followed by a second step assembly using MIRA provided effective assembly of the physical map contig, resulting in 193 contigs with aN50 value of 13,123bp.