Given:
- Ancestral DNA paired end reads (Serratia)
- paired end reads of 5 replicates from 4 treatment groups (total of 20 replicates)
- genome assembly created from ancestral DNA pair end reads
Tasks:
1. Fix Assembly by looking for contaminants
a. Align assembly against reference genome using MUMmer. Find set of sequences that align to the reference and set of sequences that did not align to the reference.
b. Take set unaligned sequences and align to a plasmids database using BLASTz or BLASTn. Take unaligned sequences and align against virulent genome. If there are still unaligned sequences then use a difference reference genome.
c. Get gene annotation of reference genome and apply coordinates from gene annotation to assembly. Make a table containing Contig ID, start, stop, length, and name of the gene.
2. Map all reads to ancestor assembly, call SNPs using SAMTools
Questions:
1. I have a script that will give me the coordinates that did align from the assembly to the reference of my choosing (I have 2 options).
With this set of coordinates, what do I do to find the plasmid contaminants given the plasmids database?
2. With the left over unaligned sequences, how do I find the virulent sequences given a virulent gene database?
3. If I am left with unmapped sequences even after looking for plasmid and virulent gene contaminants, what do I do? Use a new reference (2nd option) and try again and just go with the one with the least unmapped reads?
4. After I have figured out what portion of the unmapped reads are plasmids/virulent genes/other, what changes do I need to make to the assembly? I am very lost after this step.
My goal is to fix the assembly. I feel comfortable with cutting adapters, trimming poor quality base pairs, mapping/aligning, and SNP calling, but I really want to get the assembly and gene annotation stuff out of the way so I can actually get down to producing some results.
- Ancestral DNA paired end reads (Serratia)
- paired end reads of 5 replicates from 4 treatment groups (total of 20 replicates)
- genome assembly created from ancestral DNA pair end reads
Tasks:
1. Fix Assembly by looking for contaminants
a. Align assembly against reference genome using MUMmer. Find set of sequences that align to the reference and set of sequences that did not align to the reference.
b. Take set unaligned sequences and align to a plasmids database using BLASTz or BLASTn. Take unaligned sequences and align against virulent genome. If there are still unaligned sequences then use a difference reference genome.
c. Get gene annotation of reference genome and apply coordinates from gene annotation to assembly. Make a table containing Contig ID, start, stop, length, and name of the gene.
2. Map all reads to ancestor assembly, call SNPs using SAMTools
Questions:
1. I have a script that will give me the coordinates that did align from the assembly to the reference of my choosing (I have 2 options).
With this set of coordinates, what do I do to find the plasmid contaminants given the plasmids database?
2. With the left over unaligned sequences, how do I find the virulent sequences given a virulent gene database?
3. If I am left with unmapped sequences even after looking for plasmid and virulent gene contaminants, what do I do? Use a new reference (2nd option) and try again and just go with the one with the least unmapped reads?
4. After I have figured out what portion of the unmapped reads are plasmids/virulent genes/other, what changes do I need to make to the assembly? I am very lost after this step.
My goal is to fix the assembly. I feel comfortable with cutting adapters, trimming poor quality base pairs, mapping/aligning, and SNP calling, but I really want to get the assembly and gene annotation stuff out of the way so I can actually get down to producing some results.