Assembly
Kristoffer H. Ring
INF-BIO5121
Task 1.2 – Velvet assembly
Was planning to use VelvetOptimiser to determine the optimal kmer size,
however, this was not available on the course server.
Therefore, I took advantage of the multiprocessing capability on abel:
#Building hash tables using 39 different hash lengths, k=21-99:
bash-4.1$ velveth auto 21,99,2 -fastq -shortPaired -separate ../Sample280_1.fastq ../Sample280_2.fastq
#Submitting the velvetg-jobs to Abel (building the graphs)
./run_velvet.sh
Submit.sh
#Greping N50-values from the auto_X/log-files and plotting the results using
Python:
-bash-4.1$ python plot.py auto_*
-bash-4.1$ python plot.py auto_*
Best k = 75
Best n50 = 119377
However, k=65 and 69 might be good candidates as well.
K=75 might be a good kmer size.
The following commands were used to create assembly asm1:
-bash-4.1$ velveth asm1 75 -fastq -shortPaired -separate \
../Sample280_1.fastq ../Sample280_2.fastq \
-bash-4.1$ velvetg asm1 -exp_cov auto -cov_cutoff auto
Ran assemblathon_stat.pl on the asm1 to judge the assembly:
bash-4.1$ assemblathon_stat.pl -s 5.4 contigs.fa > metrics_asm1.txt
Most relevant metrics:
Task 1.3 – Velvet assembly with mate pair reads
Used the same approach as for the pair end reads (task 1.2)
#Building the hash indices using velveth for k=21-99:
bash-4.1$ velveth asm 21,99,2 -fastq -shortPaired -separate \
../Sample280_1.fastq ../Sample280_2.fastq \
-shortPaired2 -separate -fastq \
../TY2482_6kb_1_50x_RC.fq ../TY2482_6kb_2_50x_RC.fq
#Submitting the velvetg-jobs to Abel:
./run_velvet.sh
Submit.sh
K=45, N50=2,876,052
K=45 stands out as the best kmer size.
The following commands were used to create assembly asm2:
1.3-bash-4.1$ velveth asm2 45 -fastq -shortPaired -separate \
../Sample280_1.fastq ../Sample280_2.fastq \
-shortPaired2 -separate -fastq \
../TY2482_6kb_1_50x_RC.fq
../TY2482_6kb_2_50x_RC.fq
1.3-bash-4.1$ velvetg asm2 -exp_cov auto -cov_cutoff auto -shortMatePaired2 yes
Ran assemblathon_stat.pl on the asm2 to judge the assembly:
bash-4.1$ assemblathon_stat.pl -s 5.4 contigs.fa > metrics_asm2.txt
bash-4.1$ less metrics_asm2.txt
Most relevant Assemblathon metrics for asm2:
Insert size and standard deviation
The output from velvetg showed:
[276.666654] Paired-end library 1 has length: 444, sample standard deviation: 33
[276.850824] Paired-end library 2 has length: 6199, sample standard deviation: 777
Compared to the lecture data set, these numbers were (298, 19) and (3177, 2132) for the two
libraries respectively.
Maybe the exam assembly is better because it has a smaller standard deviation for the
mate pairs..
Task 2.1 – Spades Assembly
Ran Spades to create assembly asm3:
-bash-4.1$ module load spades
-bash-4.1$ spades.py -t 2 -k 21,29,37,43 --careful \
--pe1-1 ../Sample280_1.fastq \
--pe1-2 ../Sample280_2.fastq \
--mp1-1 ../TY2482_6kb_1_50x_RC.fq \
--mp1-2 ../TY2482_6kb_2_50x_RC.fq \
--mp1-fr -o asm3
Ran assemblathon_stat.pl on the asm2 to judge the assembly:
bash-4.1$ assemblathon_stat.pl -s 5.4 scaffolds.fasta > metrics_spades.txt
bash-4.1$ less metrics_spades.txt
Most relevant Assemblathon metrics for Spades assembly, asm3:
Task 3.1 – Mapping
Indexing the asm2 (Velvet) assembly using bwa:
-bash-4.1$ cd asm2
-bash-4.1$ module load bwa
-bash-4.1$ bwa index -a bwtsw contigs.fa
### Mapping paired end reads for asm2:
bwa mem -t 2 ../contigs.fa \
../../../Sample280_1.fastq \
../../../Sample280_2.fastq \
| samtools view -buS - | samtools sort - map_pe.sorted
## Mapping mate pairs for asm2:
bwa mem -t 2 ../contigs.fa \
../../../TY2482_6kb_1_50x_RC.fq \
../../../TY2482_6kb_2_50x_RC.fq \
| samtools view -buS - | samtools sort - map_mp.sorted
Task 3.1 – Mapping
Indexing the asm3 (Spades) assembly:
-bash-4.1$ cd asm3
-bash-4.1$ bwa index -a bwtsw contigs.fasta
-bash-4.1$ cd bwa
### Mapping paired end reads to asm3:
-bash-4.1$ bwa mem -t 2 ../scaffolds.fasta \
../../../Sample280_1.fastq \
../../../Sample280_2.fastq \
| samtools view -buS - | samtools sort - map_pe.sorted
-bash-4.1$ samtools index map_pe.sorted.bam
## Mapping mate pairs for to asm3:
-bash-4.1$ bwa mem -t 2 ../scaffolds.fasta \
../../../TY2482_6kb_1_50x_RC.fq \
../../../TY2482_6kb_2_50x_RC.fq \
| samtools view -buS - | samtools sort - map_mp.sorted
-bash-4.1$ samtools index map_mp.sorted.bam
IPython notebook plots of the insert size distribution for the asm3 assembly
Task 3.2 REAPR analysis for the asm2 and asm3 assemblies
REAPR analysis asm2 (Velvet)
-bash-4.1$ cd asm2
-bash-4.1$module load reapr
-bash-4.1$ reapr pipeline contigs.fa bwa/map_mp.sorted.bam reapr_out > reapr.out
#Replacing all spaces with in 03.score.errors.gff.gz ‘_’:
-bash-4.1$ zcat reapr_out/03.score.errors.gff.gz |sed 's/ /_/g' > 03.score.errors_nospaces.gff
##REAPR analysis asm3 (Spades)
-bash-4.1$ cd asm3
-bash-4.1$ reapr pipeline scaffolds.fasta bwa/map_mp.sorted.bam reapr_out > reapr.out
#Replacing all spaces with in 03.score.errors.gff.gz ‘_’:
-bash-4.1$ zcat reapr_out/03.score.errors.gff.gz |sed 's/ /_/g' > 03.score.errors_nospaces.gff
REAPER results
From 05.summary.report.txt for asm2
From 05.summary.report.txt for asm3
REAPR made 9 breaks in asm2 and 2 in asm3
Fragment Coverage Distribution (FCD) failure over gap in asm2
Fragment Coverage Distribution (FCD) failure over gap in asm2
Fragment Coverage Distribution (FCD) failure over gap in asm3
Conclution
The Spades assembly (asm3) might be better:
• Fewer and larger scaffolds and contigs
• Total scaffold length in agreement with known genome size (100.3%)
• REAPR identified less FCD failures over gaps (breaks).
Asseblathon stats: