Genomic Tech's COVID sequencing
For more info please see:
Full documentation and tutorial
This is a living git, that will change over time as we test out various methods. We are mostly using a 14 primer scheme, which requires some changes to the artic protocol
Also included are some handy commands for running analyses on multiple barcodes at once. We are doing both nanopolish and medaka for all runs. So far, so good.
Please let me know if anything is unclear, or you have suggestions or questions.
Working from the original artic-network protocol, we need TWO folders of files.
The first, is the protocol folder, in this case protocols/Eden/V1
The second, is the schemes folder, in this case protocols/Eden/schemes/nCoV-2019/V1
The First, is used with RAMPART in order to align and display the data with your custom primer set.
The second, is used with the artic bioinformatic analysis SOP after sequencing.
If the reference is the same as used here, then you can simply copy
genome.json
references.fasta
to your new protocol folder. Otherwise you will need to build these yourself.
(shoot me a message and I can help)
for primers.json, copy the main structure, but replace the values with your amplicon [start,stop] positions
"amplicons": [
[31, 2592],
[1876, 4450],
[4295, 6873],
...
Notice that each primer is overlapping a bit here, so you are placing them as A/B/A/B or 1/2/1/2...
The protocol.json is where you can place metadata for the run. I simply use this to name it. Fill in as you please
Again, if the reference is the same, nothing needs to be changed for
nCoV-2019.reference.fasta file
Then, if your primer bed file looks like This
MN908947.3 31 2592 A1
MN908947.3 1876 4450 B1
MN908947.3 4295 6873 A2
MN908947.3 6287 8851 B2
MN908947.3 8596 11074 A3
MN908947.3 10363 12802 B3
MN908947.3 12711 15246 A4
MN908947.3 14546 17152 B4
MN908947.3 16847 19278 A5
MN908947.3 18897 21455 B5
MN908947.3 21358 23847 A6
MN908947.3 23123 25673 B6
MN908947.3 25602 28172 A7
MN908947.3 27447 29866 B7
Then your nCoV-2019.scheme.bed looks like this:
MN908947.3 31 54 nCoV-2019_1_LEFT nCoV-2019_1
MN908947.3 2569 2592 nCoV-2019_1_RIGHT nCoV-2019_1
MN908947.3 1876 1897 nCoV-2019_2_LEFT nCoV-2019_2
MN908947.3 4429 4450 nCoV-2019_2_RIGHT nCoV-2019_2
MN908947.3 4295 4321 nCoV-2019_3_LEFT nCoV-2019_1
MN908947.3 6847 6873 nCoV-2019_3_RIGHT nCoV-2019_1
MN908947.3 6287 6310 nCoV-2019_4_LEFT nCoV-2019_2
MN908947.3 8828 8851 nCoV-2019_4_RIGHT nCoV-2019_2
...
Where you count up each pair in the 4th column, and 1 and 2 stand in for A and B in the 5th column.
(Let me know if this isn't clear, or you have a better way of explaining it)
RAMPART version in artic protocol doesn't have remote browser fix Use RAMPART install for v1.1
NB01, NB02, etc...
rampart --verbose --protocol /home/grid/SARS-CoV-2_GTG/protocols/Eden/V1/ --ports 3000 3001 --basecalledPath /data/name/name/20200330_0557_X2_ACW369_55e7e4ce/fastq_pass/
BC01, BC02, etc...
rampart --verbose --protocol /home/grid/SARS-CoV-2_GTG/protocols/Eden/V1/ --ports 5000 5001 --basecalledPath /data/name/name/20200401_0106_X5_ACL109_433d9d25/fastq_pass/ --annotationOptions require_two_barcodes="False" barcode_set="rapid"
conda activate artic-ncov2019
mkdir asm
cd asm
artic gather --min-length 2300 --max-length 2700 --prefix name --directory /data/name/name/20200330_0557_X2_ACW369_55e7e4ce/
artic demultiplex --threads 4 name_fastq_pass.fastq
cd ..
for bc in {01..09}; do mkdir barcode${bc}; cd barcode${bc}; artic minion --normalise 200 --threads 4 --scheme-directory ~/SARS-CoV-2_GTG/protocols/Eden/schemes --read-file ../asm/name_fastq_pass-NB${bc}.fastq --fast5-directory ../fast5_pass --sequencing-summary ../asm/name_sequencing_summary.txt nCoV-2019/V1 name; cd ../; done
This is still in testing, use native barcodes
porechop --verbosity 2 --untrimmed -i "name_fastq_pass.fastq" -b ./ --rapid_barcodes --discard_middle --barcode_threshold 80 --threads 4 --check_reads 10000 --barcode_diff 5 > name_fastq_pass.fastq.demultiplexreport.txt
for file in BC*.fastq; do mv $file name_fastq_pass-${file}; done
conda activate artic-ncov2019-medaka
mkdir asm
# directory is an example, but should point to the folder with the sequencing_summary.txt
artic gather --min-length 2300 --max-length 2700 --prefix name --directory /data/name/name/20200330_0557_X2_ACW369_55e7e4ce/
artic demultiplex --threads 4 name_fastq_pass.fastq
cd ..
for bc in {01..09}; do mkdir barcode${bc}; cd barcode${bc}; artic minion --minimap2 --medaka --normalise 200 --threads 4 --scheme-directory ~/SARS-CoV-2_GTG/protocols/Eden/schemes --read-file ../asm/name_fastq_pass-NB${bc}.fastq nCoV-2019/V1 name; cd ../; done
Checking the reference used is the same, and if not, how they differ
pip3 install edlib
python3 compare_ref.py -r1 base_reference.fasta -r2 new_reference.fasta
New commands - experimental
artic gather --min-length 300 --max-length 2700 --prefix PQPR071978 --directory /data/PQPR071978/20200527_0703_3D_PAE51904_eb3de804/ --fast5-directory /data/PQPR071978/20200527_0703_3D_PAE51904_eb3de804/
# Native
artic demultiplex --threads 32 PQPR071978_fastq_pass.fastq
# Rapid
porechop --verbosity 2 --untrimmed -i "PQPR071978_fastq_pass.fastq" -b ./ --rapid_barcodes --discard_middle --barcode_threshold 80 --threads 32 --check_reads 10000 --barcode_diff 5 > PQPR071978_fastq_pass.fastq.demultiplexreport.txt
# Nanopolish
RUN=PQPR071978 && for bc in {01..12}; do mkdir barcode${bc}; cd barcode${bc}; artic minion --normalise 200 --threads 32 --scheme-directory /directflow/KCCGGenometechTemp/projects/jamfer/SARS-CoV-2/SARS-CoV-2_GTG/protocols/Eden/schemes --read-file ../../base/${RUN}_fastq_pass-BC${bc}.fastq --fast5-directory ../../${RUN}/*/fast5_pass --sequencing-summary ../../${RUN}_summary.txt nCoV-2019/V1 ${RUN}; cd ../; done
# Medaka
RUN=PQPR071978 && for bc in {01..12}; do mkdir barcode${bc}; cd barcode${bc}; artic minion --minimap2 --medaka --normalise 200 --threads 32 --scheme-directory /directflow/KCCGGenometechTemp/projects/jamfer/SARS-CoV-2/SARS-CoV-2_GTG/protocols/Eden/schemes --read-file ../../base/${RUN}_fastq_pass-BC${bc}.fastq nCoV-2019/V1 ${RUN}; cd ../; done
# dRNA - VERY experimental
rampart --verbose --protocol /home/prom/SARS-CoV-2_GTG/protocols/Eden/RNA --ports 6010 6011 --basecalledPath /data/<project>/*/*/fastq_pass/
#Running multiple samples
#for RUN in $(cut -f2 ./list_nat_comms_multiples.tsv | sort | uniq); do mkdir ${RUN}; cd ${RUN}; ls ../../big_one/${RUN}; NUM_JOBS=$(find ../../big_one/${RUN} -type f -name "*.fast5" | wc -l); echo ${NUM_JOBS}; qsub -V -t 1-${NUM_JOBS} /directflow/KCCGGenometechTemp/projects/jamfer/scripts/basecalling.sge ../../big_one/${RUN} dna_r9.4.1_450bps_hac.cfg; cd ..; done
for RUN in $(cut -f2 ./list_nat_comms_singles.tsv | sort | uniq | grep GQ); do echo ${RUN}; cd ${RUN}; mkdir base medaka nanopolish; cd base; artic gather --min-length 300 --max-length 2700 --prefix ${RUN} --directory ../${RUN}/ --fast5-directory ../../../big_one/${RUN}/${RUN}/*/ ; ls -la; porechop --verbosity 2 --untrimmed -i "${RUN}_fastq_pass.fastq" -b ./ --rapid_barcodes --discard_middle --barcode_threshold 80 --threads 32 --check_reads 10000 --barcode_diff 5 > ${RUN}_fastq_pass.fastq.demultiplexreport.txt; for f in BC*.fastq; do mv $f ${RUN}_fastq_pass-${f}; ls -la; done; cd ../nanopolish; for BC in $(grep ${RUN} ../../list_nat_comms_singles.tsv | cut -f3 | cut -d 'B' -f 2); do NAME=$(grep ${RUN} ../../list_nat_comms_singles.tsv | grep RB${BC} | cut -f1); mkdir ${NAME}_${RUN}_BC${BC}_nanopolish; cd ${NAME}_${RUN}_BC${BC}_nanopolish; artic minion --normalise 200 --threads 32 --scheme-directory /directflow/KCCGGenometechTemp/projects/jamfer/SARS-CoV-2/SARS-CoV-2_GTG/protocols/Eden/schemes --read-file ../../base/${RUN}_fastq_pass-BC${BC}.fastq --fast5-directory ../../../../big_one/${RUN}/${RUN}/*/fast5_pass --sequencing-summary ../../${RUN}/${RUN}_summary.txt nCoV-2019/V1 ${NAME}_${RUN}_BC${BC}_nanopolish; cd .. ; done ; cd ../..; done
for RUN in $(cut -f2 ./list_nat_comms_singles.tsv | sort | uniq | grep GQ); do echo ${RUN}; cd ${RUN}; cd medaka; for BC in $(grep ${RUN} ../../list_nat_comms_singles.tsv | cut -f3 | cut -d 'B' -f 2); do NAME=$(grep ${RUN} ../../list_nat_comms_singles.tsv | grep RB${BC} | cut -f1); mkdir ${NAME}_${RUN}_BC${BC}_medaka; cd ${NAME}_${RUN}_BC${BC}_medaka; artic minion --minimap2 --medaka --normalise 200 --threads 32 --scheme-directory /directflow/KCCGGenometechTemp/projects/jamfer/SARS-CoV-2/SARS-CoV-2_GTG/protocols/Eden/schemes --read-file ../../base/${RUN}_fastq_pass-BC${BC}.fastq nCoV-2019/V1 ${NAME}_${RUN}_BC${BC}_medaka; cd .. ; done ; cd ../..; done
# medaka 1.1.1
for RUN in $(cut -f2 ./list_nat_comms_singles.tsv | sort | uniq | grep GQ); do echo ${RUN}; cd ${RUN}; mkdir medaka_2; cd medaka_2; for BC in $(grep ${RUN} ../../list_nat_comms_singles.tsv | cut -f3 | cut -d 'B' -f 2); do NAME=$(grep ${RUN} ../../list_nat_comms_singles.tsv | grep RB${BC} | cut -f1); mkdir ${NAME}_${RUN}_BC${BC}_medaka_2; cd ${NAME}_${RUN}_BC${BC}_medaka_2; artic minion --minimap2 --medaka --normalise 200 --medaka-model r941_min_high_g360 --threads 32 --scheme-directory /directflow/KCCGGenometechTemp/projects/jamfer/SARS-CoV-2/SARS-CoV-2_GTG/protocols/Eden/schemes --read-file ../../base/${RUN}_fastq_pass-BC${BC}.fastq nCoV-2019/V1 ${NAME}_${RUN}_BC${BC}_medaka_2; cd .. ; done ; cd ../..; done
--medaka-model r941_prom_high_g4011
This is the joint work with
Rowena A. Bull, Thiruni N. Adikari, James M. Ferguson, Jillian M. Hammond, Igor Stevanovski, Alicia G. Beukers, Zin Naing, Malinna Yeang, Andrey Verich, Hasindu Gamaarachchi, Ki Wook Kim, Fabio Luciani, Sacha Stelzer-Braid, John-Sebastian Eden, William D. Rawlinson, Sebastiaan J. van Hal & Ira W. Deveson
See the paper here: https://www.nature.com/articles/s41467-020-20075-6