---
# System prepended metadata

title: Mouse ont assembly and pangenome

---

# Mouse ont assembly and pangenome

Assembly one sample (BXD24).


|Reads|	Yield_[bp]|	N50|	Coverage|	Max_length	|Mean_length|	Median_length	|Mean_q	|Median_q|
|----|----|---|---|---|---|---|--|--|
|  4,032,662 |	  81,375,782,055 |	  36,289 |	  30 |	  669,638 |	  20,179 |	  13,362 |	  13.34 |	  14.40 

### 1. Generate the assembly 

- I use wtdbg2 to perform an assembly of long-read nanopore sequencing data (-x ont) from a fastq file obtained by the base calling. 
- I use wtpoa-cns to polish the assembly generated in step 1.
- I use minimap2 to align the original long-read nanopore sequencing data to the polished assembly from step 2. The command use the option specify that we are mapping long reads (-ax map-ont) and that the reference genome was assembled using a long-read assembly algorithm (-r2k). The output is a BAM file, in which I filter the aligned reads to remove secondary and supplementary alignments (specified by the "-F0x900" option).

```
sbatch  -p workers -w octopus02 -c 48 --wrap 'cd /scratch && /lizardfs/flaviav/mouse_ont/assembly/as.sh'
Submitted batch job 124177
```

### 1b. Statistics on the assembly (quast)

quast.py mouse_reads.ont.wtdbg2.asm1.cns.fa.gz 
![](https://i.imgur.com/iR6yL2Z.png)


### 1c. Assembly with canu

```
sbatch  -p workers -w octopus02 -c 48 --wrap 'cd /scratch && /lizardfs/flaviav/mouse_ont/canu.sh'

submitted batch job 125166
```

### 2. Polishing the assembly with linked reads

```
 bwa-0.7.17/bwa index mouse_reads.ont.wtdbg2.asm1.cns.fa
```

```
sbatch  -p workers -w octopus05 -c 48 --wrap 'cd /scratch && /lizardfs/flaviav/mouse_ont/assembly/polish.sh' 
Submitted batch job 125066

```

- Alignment: BWA mem to align linked reads to the ONT-based assembly of the mouse genome. 
- Sorting: The resulting SAM file from the alignment step is then converted to a binary BAM file and sorted using Sambamba. 
- Variant calling: This command uses Freebayes to call variants on each contig of the ONT-based assembly of the mouse genome. 
- Concatenation: This command concatenates the variant calls from each contig into a single BCF file using BCFTOOLS. The -n and -f flags are used to specify the list of BCF files to concatenate and the reference FASTA file to use during the normalization process, respectively.
- Polishing: This command uses the polished BCF file generated in step 4 to polish the ONT-based assembly of the mouse genome. The -i, -H, and -f flags are used to specify the filter expression for variant calling.

Error in the sorting step, sambamba--> Pjotr is checking and I'll use GATK instead of sambamba for now
