Organizing the project folder
Make a main directory where all the work will be performed.
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mkdir BTspades
cd BTspades
Downloading the dataset
Make a subdirectory for the raw data files.
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mkdir 00_rawdata
Download the raw data from EBI.
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wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR209/001/SRR2093871/SRR2093871_1.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR209/001/SRR2093871/SRR2093871_2.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR209/002/SRR2093872/SRR2093872_1.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR209/002/SRR2093872/SRR2093872_2.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR209/006/SRR2093876/SRR2093876_subreads.fastq.gz
QC on the data
Illumina data
If you only have a few files to check, it is easier to start an interactive node and run them quickly in the terminal.
Start interactive node.
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salloc -N 1 -t 4:00:00
Load the fastqc module and execute fastqc for each file.
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module load fastqc
fastqc --version
FastQC v0.11.3
fastqc SRR2093871_1.fastq.gz
fastqc SRR2093871_2.fastq.gz
fastqc SRR2093872_1.fastq.gz
fastqc SRR2093872_2.fastq.gz
Results
- Fastqc results for SRR2093871_1
- Fastqc results for SRR2093871_2
- Fastqc results for SRR2093872_1
- Fastqc results for SRR2093872_2
FASTQC indicates there are no major issues with these data.
For datasets with many files see our tutorial on QC [TODO Add tutorial on QC]
PacBio or long read data
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SRR2093876_subreads.fastq.gz
Genome assembly using Spades
Change back into the main folder BTspades and make a subdirectory for the Spades assembly files
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mkdir 01_spades
cd 01_spades
spades.sub header used on XSEDE bridges
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#!/bin/bash
#SBATCH -J spades4.e%j
#SBATCH -o spades4.o%j
#SBATCH --mem 1000GB
#SBATCH -p LM
#SBATCH -N 1
#SBATCH -n 40
#SBATCH -t 48:00:00
source /usr/share/Modules/init/bash
module load spades/3.11.1
Running Spades
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module load spades
spades.py \
-k 21,33,55,77,99,127 \
-t 16 -m 1000 \
--pacbio /pylon5/mc48o5p/severin/spadesTutorial/00_rawdata/SRR2093876_subreads.fastq.gz \
--pe1-1 /pylon5/mc48o5p/severin/spadesTutorial/00_rawdata/SRR2093871_1.fastq.gz \
--pe1-2 /pylon5/mc48o5p/severin/spadesTutorial/00_rawdata/SRR2093871_2.fastq.gz \
--mp1-1 /pylon5/mc48o5p/severin/spadesTutorial/00_rawdata/SRR2093872_1.fastq.gz \
--mp1-2 /pylon5/mc48o5p/severin/spadesTutorial/00_rawdata/SRR2093872_2.fastq.gz \
--careful \
-o BT
| SPAdes | files output | from assembly |
|---|---|---|
| K21 | K33 | K55 |
| K77 | K99 | assembly_graph.fastg |
| assembly_graph_with_scaffolds.gfa | before_rr.fasta | contigs.fasta |
| contigs.paths | corrected | dataset.info |
| input_dataset.yaml | misc | mismatch_corrector |
| params.txt | scaffolds.fasta | scaffolds.paths |
| spades.log | tmp | warnings.log |
Assembly statistics
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new_Assemblathon scaffolds.fasta
---------------- Information for assembly './scaffolds.fasta' ----------------
Number of scaffolds 1334
Total size of scaffolds 6768928
Longest scaffold 395916
Shortest scaffold 100
Number of scaffolds > 1K nt 104 7.8%
Number of scaffolds > 10K nt 58 4.3%
Number of scaffolds > 100K nt 23 1.7%
Number of scaffolds > 1M nt 0 0.0%
Number of scaffolds > 10M nt 0 0.0%
Mean scaffold size 5074
Median scaffold size 306
N50 scaffold length 218044
L50 scaffold count 12
scaffold %A 31.95
scaffold %C 17.95
scaffold %G 18.09
scaffold %T 32.01
scaffold %N 0.00
scaffold %non-ACGTN 0.00
Number of scaffold non-ACGTN nt 0
Percentage of assembly in scaffolded contigs 0.0%
Percentage of assembly in unscaffolded contigs 100.0%
Average number of contigs per scaffold 1.0
Average length of break (>25 Ns) between contigs in scaffold 0