added assembly

Author: HadrienG

assembly.md

@@ -8,7 +8,9 @@ Go to ENA, and search for the run ERR486840.
 
 Download the 2 fastq files associated with the run.
 
-## Quality control?
+## Quality control
+
+Perform quality control of the data as you did in the [QC tutorial](qc.md)
 
 How many reads are in the fastq file? What is the read length?
 Does the data need trimming or other filtering? If so, do it.
@@ -20,23 +22,75 @@ Based on the expected genome size, the read length and the number of reads – w
 
 We will be using the SPAdes assembler to assemble our bacterium.
 
+```
+module load spades
+spades.py -k 21,33,55,77,99 --careful --only-assembler -1 read_1.fastq -2 read_2.fastq -o output
+```
+
 This will produce a series of outputs. The scaffolds will be in fasta format.
 
 How well does the assembly total consensus size and coverage correspond to your earlier estimation?
 What is the N50 of the assembly? What does this mean?
 How many contigs in total did the assembly produce?
 How many contigs longer than 500bp? What is the N50 of those contigs only?
 
-Perform more assemblies with the following options:
+Perform another assembly with the following options:
+
+Use the raw reads (no trimming, but with adapters removed), wthout the --only-assembler option.
+
+If you have time, try out the following genome assemblers:
 
-Raw reads (no trimming, but with adapters removed), let spades do the qc.
+
+* MaSurCa
+* Ray
 
 ## Comparing assemblies
 
-quast
+QUAST is a software evaluating the quality of genome assemblies by computing various metrics, including
+
+* N50, length for which the collection of all contigs of that length or longer covers at least 50% of assembly length
+* NG50, where length of the reference genome is being covered
+* NA50 and NGA50, where aligned blocks instead of contigs are taken
+* misassemblies, misassembled and unaligned contigs or contigs bases
+* genes and operons covered
+
+To run Quast:
+
+```
+module load quast
+quast.py assembly1.fasta assembly2.fasta ... -R reference.fasta -G reference.gff
+```
+
+Quast will produce a pdf in the `quast_results` directory. Download it on your computer and take a look. Which assembly is better?
+
+## Fixing misassemblies
+
+Pilon is a software tool which can be used to automatically improve draft assemblies. It attempts to make improvements to the input genome, including:
+
+* Single base differences
+* Small Indels
+* Larger Indels or block substitution events
+* Gap filling
+* Identification of local misassemblies, including optional opening of new gaps
+
+Pilon then outputs a FASTA file containing an improved representation of the genome from the read data and an optional VCF file detailing variation seen between the read data and the input genome.
+
+You can read how Pilon works in detail [here](https://github.com/broadinstitute/pilon/wiki/Methods-of-Operation)
+
+Before running Pilon itself, you have to map your reads back to the assembly!
+
+```
+bowtie2-build $assembly $output/index
+(bowtie2 -x $output/index -1 $r1 -2 $r2 | samtools view -bS -o $mapping - ) 2> bowtie.err
+samtools sort $mapping $mapping.sorted
+samtools index $mapping.sorted.bam
+```
 
-### Comparing to the reference?
+Run Pilon with the following command:
 
-Get the reference fasta here
+```
+module load pilon
+pilon --genome $assembly --frags $mapping.sorted.bam --output $output
+```
 
-## Fixing misassemblies?
+Once Pilon is finished running, compare the new assembly with the old one using Quast!