**Please submit bugs under the 'Issues' tab. Send comments, corrections, or suggestions to RLN0005@auburn.edu
For a video walkthrough, please see: https://www.youtube.com/watch?v=Uj9rs9EAMu8&t=17s
This script was initially intended to be used on sequence data from a circular haploid plasmid genome of approximately 37 kb that was sequenced using Oxford Nanopore's (ONT) minION device. It was intended to be used as a quick reference to determine if CRISPR-modified plasmid genomes were accurately constructed in the lab prior to transfection. Visualization of assmeblies using Bandage and variants using IGV allows regions of dissimilarity to be further investigated.
This script (minION_align.sh) is designed to use sequence data from a Nanopore minION (or any long-read sequence data) specifically outputted from the minKNOW program (in .fastq format). The script will combine all .fastq files for an individual barcode into a single .fastq file, filter the reads using NanoFilt, map reads in a few different ways with different packages -- including de novo assembly using Shasta, de novo assembly using Minimap2/Miniasm, and assembly using a reference sequence using Minimap2/Miniasm. Racon will be used for error correction and to generate a consensus sequence for each of the assemblies.
Some difficulties encountered that are specific to the aims of this project include: 1) draft assembly/consensus sequence generation for relatively small CIRCULAR plasmids (some reads span the entire plasmid and more) and 2) difficulty in SNP/indel variant calling using long-read HAPLOID sequence data. As new tools are developed and pre-existing tools are improved, this script/repository will be updated.
In its current state, this script does not call variants (SNPs/indels) reliably; this part of the script is still being debugged. It does, however, generate multiple consensus files and draft assemblies that are generated using several different tools.
There are 5 general steps in this script: 1) Combine & Filter 2) Align/Map 3) Generate Consensus 4) Variant Call 5) Visualize Assemblies/Variants. The 5th step is not executed by the script itself (see info below for options to visualize results).
Use CL tools (such as grep) to extract barcodes for samples so that they can be processed individually (for example, different reference sequences for barcodes 1 and 2). Combine all fastq files for a given barcode into a single fastq file.
These reads are already labeled 'pass' -- I think they have already been filtered to an extent. In the future, I plan to implement Guppy as a basecaller in this script.
Using NanoFilt with standard/fixed filtering conditions. These parameters can be adjusted within the script if desired.
NanoFilt -l 500 -q 12 --headcrop 50 < *_all.fastq > trim.fastq
-l 500 option filters based on average sequence length of 500 bp
-q 12 option filters based on average quality score of 12
--headcrop 50 filters by removing first 50bp of sequence
Align reads with a reference sequence using minimap2 to align with reference sequence (used for ONT genomic reads).
minimap2 -x map-ont *_ref.fasta trim.fastq | gzip -1 > map_minimap_ref.paf.gz
Assemble Untigs: miniasm -f trim.fastq map_minimap_ref.paf.gz > map_minimap_ref.gfa
Generate consensus by converting gfa to fasta: awk `/^S/{print ">"$2"\n"$3}' map_minimap_ref.gfa > map_minimap_ref.fasta
Map Trimmed Reads onto Miniasm Assembly: minimap2 map_minimap_ref.fasta trim.fastq > map_minimap_ref.racon.paf
The 'map_minimap_ref.racon.paf' file will be used in the error correction step using Racon below.
This will generate a new subfolder called "ShastaRun" which will contain many files, including Assembly.gfa and Assembly.fasta.
shasta-Linux-0.7.0 --input *_all.fastq
Error Correction using Racon can be applied to the Shasta mapped file -- to compare consensus assemblies generated using different alignment programs (Shasta vs. Minimap2/Miniasm)
Map Reads Onto Themselves -- Identifies Overlaps (untigs): minimap2 -x ava-ont trim.fastq trim.fastq | gzip -1 > overlaps.paf.gz
Assemble Untigs: miniasm -f trim.fastq overlaps.paf.gz > untigs.gfa
Generate consensus by converting gfa to fasta: awk `/^S/{print ">"$2"\n"$3}' untigs.gfa > untigs.fasta
Map Trimmed Reads onto Miniasm Untigs Assembly: minimap2 untigs.fasta trim.fastq > untigs.racon.paf
The 'untigs.racon.paf' file will be used in the error correction step using Racon below.
Racon was developed to complement minimap2/miniasm pipeline but can be used for any long-read assembly. It is used to correct draft assemblies.
Build Consensus Using Trimmed Reads and Minimap/Untigs.Racon Assembly (from de novo assembly with Minimap):
racon trim.fastq untigs.racon.paf untigs.fasta > map_minimap_denovo.racon.consensus.fasta
Build Consensus Using Trimmed Reads and Contigs/Shasta Assembly (from de novo assembly with Shasta):
racon trim.fastq overlaps.paf.gz ShastaRun/Assembly.fasta > map_shasta_denovo.racon.consensus.fasta
Build Consensus Using Trimmed Reads and Minimap Assembly (from assembly using reference with Minimap):
racon trim.fastq map_minimap_ref.racon.paf map_minimap_ref.fasta > map_minimap_ref.racon.consensus.fasta
Further iterations with Racon can be utilized (but are not included in this script currently); for example:
minimap2 map_minimap_denovo.racon.consensus.fasta trim.fastq > map_minimap_denovo.racon2.paf
racon trim.fastq map_minimap_denovo.racon2.paf map_minimap_denovo.racon.consensus.fasta > map_minimap_ref.racon2.consensus.fasta
- Assembly from reference using Minimap2 (map_minimap_ref.gfa)
- De Novo assembly using Minimap2 (map_minimap_denovo.gfa)
- De Novo assembly using Shasta (/ShastaRun/Assembly.gfa)
- Assembly from reference using Minimap2 followed by error correction/consensus with Racon (map_minimap_denovo.racon.consensus.fasta)
- De Novo assembly using Minimap2 followed by error correction/consensus with Racon (map_shasta_denovo.racon.consensus.fasta)
- De Novo assembly using Shasta followed by error correction/consensus with Racon (map_minimap_ref.racon.consensus.fasta)
It has been difficult to find a reliable SNP/indel variant caller for long read data on a haploid genome. Medaka has a command medaka_haploid_variant that is supposed to be used to generate variants on haploid sequences, but there are some bugs in their code. See below.
medaka_haploid_variant -t 2 trim.fastq *_ref.fasta
BUG IN MEDAKA CODE -- the output of medaka_haploid_variant includes a vcf file (consensus_to_ref.vcf) but this file is generated with blanks (whitespace) in the "INFO" column -- which is required to be populated to be visualized in IGV. This has already been reported to medaka team (see nanoporetech/medaka#286). I attempted to overcome this with the following: medaka tools annotate consensus_to_ref.vcf *_ref.fasta mapped.sorted.bam variants.annotated.vcf This will output a file variants.annotated.vcf which can be uploaded into IGV.
I believe medaka_haploid_variant includes the medaka_consensus step within it, but this is not directly clear from the manual.
Type igv on the command line to open the IGV program -- upload files directly.
Upload the reference fasta file using Genomes --> upload from file.
Upload the vcf file using File --> upload from file. Upload the sorted bam file (mapped.sorted.bam) using File --> upload from file.
Type Bandage on the command line to open the Bandage GUI -- assemblies (gtf files) can be uploaded & visualized directly
Sniffles is used for structural variants, which is not the intention of this current project. But here's some info on it:
Using Sniffles (https://github.com/fritzsedlazeck/Sniffles)
sniffles -m mapped.sorted.bam -v variants.vcf
-m option is used to indicate the mapped bam file name
-v option is used to indicate the output vcf file name
Canu is another popular assembly package. This was attempted using this dataset, but the program ran for approximately 12+ hours (on a standard laptop, not HPC) and was still running. It was purposefully killed (and otherwise might have taken days to complete the run). This option should be explored using HPC.
canu -p output genomeSize=37.8k -corMemory=3 -corThreads=3 -redMemory=3 -redThreads=2 -oeaMemory=3 -oeaThreads=2 -nanopore-raw *_all.fastq
Since we are not expecting major variations between the sequenced data and the reference sequence, MUMmer (https://github.com/mummer4/mummer) was installed and dnadiff was attempted using dnadiff <reference.fasta> <consensus.fasta> but repeatedly got "ERROR: Failed to run show-snps, aborting." Consensus quality can also be explored using dnadiff. This tool should be further explored for the purposes of this script.
Graphmap (https://github.com/isovic/graphmap) tool to map/align long reads to a reference sequence. Unfortunately I was unable to get this package successfully installed on my machine, but this could be another potential mapping tool to explore.
- NanoFilt (https://github.com/wdecoster/nanofilt)
- Minimap2 (https://github.com/lh3/minimap2)
- IGV (https://github.com/igvteam/igv)
- Bandage (https://github.com/rrwick/Bandage)
- Racon (https://github.com/isovic/racon)
- Shasta (https://github.com/chanzuckerberg/shasta)
- Medaka (https://github.com/nanoporetech/medaka)
- Script is written in bash and can be executed on the command line using 'sh minION_align.sh'
- Script should be executed within a directory titled 'minION_align'.
- Sequence data files (fastq) should be contained in a subdirectory titled 'data'. (minION_align/data)
- A reference genome in fasta format should contain 'ref.fasta' in the file name and should be contained in the 'minION_align' main directory.
- All output files will be deposited in the 'minION_align' main directory unless otherwise specified.
- The fastq file combining all fastq files for an individual barcode will include the file suffix 'all.fastq' and will be deposited in the 'minION_align' main directory.