The shorter sequence read length, higher base-call error rate, non-uniform coverage and platform-specific artifacts of next-generation sequencing (NGS) technologies hinder the de novo genome assembly. We developed ngsShoRT (next-generation sequencing Short Reads Trimmer), a flexible and comprehensive open source software package written in Perl that implements novel algorithms as well as other commonly used pre-processing algorithms from the literature to process Illumina short read sequences for downstream data analyses. We compared the effects of different pre-processing algorithms/methods on the de novo assembly of C. elegans genome by measuring the assembly contiguity and correctness. Our experiments show that removing reads with ambiguous "N" bases and adapter sequences, and trimming of 3′-ends of reads using our novel quality score-based trimming algorithms improved the assembly quality. The source code of ngsShoRT is freely available and can be easily incorporated as a pre-processing step for genome/transcriptome assembly pipeline.