Hello everyone,

this is my first question here, and I am still quite new with this topic. I need to assemble short reads guided (or not) by a reference sequence using Phrap.

I have a FASTA file with 50bp paired-end reads (I also have it in SAM, BAM, and FASTQ formats) mapping to a full reference sequence I have in FASTA format as well. I obtained my read maps with Bowtie2 and samtools.

I explicitly want to use Phrap to obtain a full-length assemble of the reads and compare it to the reference via a pairwise alignment with Needle. I want to do it using the reference as guide, and not using it as well.

I have been sent the Phred and Phrap programs, but I am quite lost. I have tried Phrap alone with no quality file, but I get many short contigs instead of one long one.

I understand I should follow the whole Phred -> Phd2fasta -> CrossMatch -> Phrap protocol, but I do not seem to find my way around it. It seems Phred uses a chromatogram file as input, but I do not know how to obtain it.

So my question is how should I follow the Phred/Phrap protocol starting with a FASTA file (SAM, BAM, or FASTQ) with 50bp reads mapping a reference FASTA file, as inputs? I want to obtain a contig that spans the full length of the reference (using the reference and not using it as input).

Thanks a lot!