denovo illumina and isoseq txomes

05-transcriptomes/README.md

+# Developmental transcriptomes
+
+We obtained developmental transcriptomes from different stages of *Pycnogonum litorale* development.
+Staging was done after molts, where possible. The following stages were sampled:
+
+- embryonic stage 3-4
+- instar I-protonymphon
+- instar II
+- instar III
+- instar IV
+- instar V
+- instar VI
+- juvenile I
+- subadult
+
+The samples were sequenced on the Illumina NovaSeq platform, with 150bp paired-end reads.
+
+We also obtained short-read sequencing data from our collaborators, though at lower sequencing
+coverage:
+
+- zygote
+- early cleavage
+- embryo 0-1
+- embryo 3-5
+- embryo 9-10
+- mixed instar II-V
+
+We also generated long-read HiFi datasete from mixed developmental stages of _Pycnogonum litorale_
+as part of a multiplexed Iso-seq run, together samples from two further species.
+
+### Short-read de novo transcriptome assembly
+
+We assembled the deeply sequenced short-read transcriptomes (embryonic stage 3-4, instar
+I-protonymphon, instar II, instar III, instar IV, instar V, instar VI, juvenile I, subadult) with
+Trinity.
+
+- [`assemble-all-sep.sh`](assemble-all-sep.sh): main script, submits single scripts
+- [`assemble-single.sh`](assemble-single.sh): commands to assemble a single dataset
+
+### Iso-seq processing
+
+We followed the protocol for Iso-seq processing as [described by the
+manufacturer](https://isoseq.how/clustering/)
+
+- Produce [consensus](isoseq-01-consensus.sh) HiFi reads from the ZMWs.
+- Remove primers and identify barcodes with [`lima`](isoseq-02-lima.sh) 
+- Trim poly(A) tails and remove concatemers with [`refine`](isoseq-03-refine.sh) 
+- [Cluster](isoseq-04-cluster.sh) the reads into isoforms
+- [Align](isoseq-05-align.sh) to the genome
+
+### Evaluation
+
+We [evaluated](eval-busco.sh) the de-novo assemblies with BUSCO v5.2.0, using the arthropoda_odb10
+database.
+
+| stage                 | complete | single | duplicated | fragmented | missing | total |
+|-----------------------|----------|--------|------------|------------|---------|-------|
+| embryonic_stage3-4    | 98.1%    | 44.7%  | 53.4%      | 0.7%       | 1.2%    | 1013  |
+| instar_I-protonymphon | 98.8%    | 47.3%  | 51.5%      | 0.7%       | 0.5%    | 1013  |
+| instar_II             | 99.4%    | 42.3%  | 57.1%      | 0.3%       | 0.3%    | 1013  |
+| instar_III            | 99.2%    | 43.5%  | 55.7%      | 0.4%       | 0.4%    | 1013  |
+| instar_IV             | 99.2%    | 37.3%  | 61.9%      | 0.4%       | 0.4%    | 1013  |
+| instar_V              | 99.3%    | 44.7%  | 54.6%      | 0.4%       | 0.3%    | 1013  |
+| instar_VI             | 99.0%    | 47.0%  | 52.0%      | 0.5%       | 0.5%    | 1013  |
+| juvenile_I            | 99.2%    | 44.1%  | 55.1%      | 0.4%       | 0.4%    | 1013  |
+| subadult              | 99.3%    | 47.5%  | 51.8%      | 0.2%       | 0.5%    | 1013  |

05-transcriptomes/assemble-all-sep.sh

+#!/usr/bin/env bash
+
+# A script to submit the transcriptome of each developmental stage to the cluster to be assembled
+# with Trinity. I will then merge the assemblies into a single one.
+
+
+INPUT="/scratch/zoology/pycnogonum/HTT33DSX5_4_R15615_20230713/demultiplexed"
+BASE="/scratch/zoology/pycnogonum/transcriptome/dev_timepoints"
+SCRIPT="/home/user/papadopoulos/repos/pycno-seq/dev_transcriptomes/assemble-single.sh"
+
+
+sbatch -J pycno-trinity-protonymphon_instar1 $SCRIPT "$INPUT"/235253/235253_S47_L004_R1_001.fastq.gz "$INPUT"/235253/235253_S47_L004_R2_001.fastq.gz "${BASE}/235253_trinity_protonymphon_instar1/"
+sbatch -J pycno-trinity-instar6 $SCRIPT "$INPUT"/235254/235254_S48_L004_R1_001.fastq.gz "$INPUT"/235254/235254_S48_L004_R2_001.fastq.gz "${BASE}/235254_trinity_instar6/"
+sbatch -J pycno-trinity-embryonic_stage3 $SCRIPT "$INPUT"/235255/235255_S49_L004_R1_001.fastq.gz "$INPUT"/235255/235255_S49_L004_R2_001.fastq.gz "${BASE}/235255_trinity_embryonic_stage3-4/"
+sbatch -J pycno-trinity-instar2 $SCRIPT "$INPUT"/235256/235256_S50_L004_R1_001.fastq.gz "$INPUT"/235256/235256_S50_L004_R2_001.fastq.gz "${BASE}/235256_trinity_instar2/"
+sbatch -J pycno-trinity-instar3 $SCRIPT "$INPUT"/235257/235257_S51_L004_R1_001.fastq.gz "$INPUT"/235257/235257_S51_L004_R2_001.fastq.gz "${BASE}/235257_trinity_instar3/"
+sbatch -J pycno-trinity-instar4 $SCRIPT "$INPUT"/235258/235258_S52_L004_R1_001.fastq.gz "$INPUT"/235258/235258_S52_L004_R2_001.fastq.gz "${BASE}/235258_trinity_instar4/"
+sbatch -J pycno-trinity-instar5 $SCRIPT "$INPUT"/235259/235259_S53_L004_R1_001.fastq.gz "$INPUT"/235259/235259_S53_L004_R2_001.fastq.gz "${BASE}/235259_trinity_instar5/"
+sbatch -J pycno-trinity-juvenile1 $SCRIPT "$INPUT"/235260/235260_S54_L004_R1_001.fastq.gz "$INPUT"/235260/235260_S54_L004_R2_001.fastq.gz "${BASE}/235260_trinity_juvenile1/"
+sbatch -J pycno-trinity-subadult $SCRIPT "$INPUT"/235261/235261_S55_L004_R1_001.fastq.gz "$INPUT"/235261/235261_S55_L004_R2_001.fastq.gz "${BASE}/235261_trinity_subadult/"
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05-transcriptomes/assemble-single.sh

+#!/usr/bin/env bash
+#
+#SBATCH --cpus-per-task=16
+#SBATCH --mem=50G
+#SBATCH --time=48:00:00
+#SBATCH --mail-type=ALL
+#SBATCH --mail-user=nikolaos.papadopoulos@univie.ac.at
+#SBATCH --output=/lisc/user/papadopoulos/log/%x-%j.out
+#SBATCH --error=/lisc/user/papadopoulos/log/%x-%j.err
+
+module load assembly/trinityrnaseq/2.15.1
+
+R1=$1
+R2=$2
+ASSEMBLY=$3
+
+mkdir -p "$ASSEMBLY" || exit 1
+
+Trinity \
+ --seqType fq \
+ --max_memory 50G \
+ --trimmomatic \
+ --no_salmon \
+ --output "$ASSEMBLY" \
+ --CPU 16 \
+ --left "$R1" \
+ --right "$R2"
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05-transcriptomes/eval-busco.sh

+#!/usr/bin/env bash
+
+# put de novo transcriptomes in an array
+BASE="/scratch/zoology/pycnogonum/transcriptome/development"
+
+embryonic_stage34=$BASE/embryonic_stage3-4
+instarI_protonymphon=$BASE/instarI-protonymphon
+instar_II=$BASE/instar_II
+instar_III=$BASE/instar_III
+instar_IV=$BASE/instar_IV
+instar_V=$BASE/instar_V
+instar_VI=$BASE/instar_VI
+juvenile_I=$BASE/juvenile_I
+subadult=$BASE/subadult
+
+TXOMES=("$embryonic_stage34" "$instarI_protonymphon" "$instar_II" "$instar_III" "$instar_IV" "$instar_V" "$instar_VI" "$juvenile_I" "$subadult")
+
+# define BUSCO script
+BUSCO=/lisc/user/papadopoulos/repos/pycno-seq/nanopore/eval-busco.sh
+
+# loop over the assemblies and submit the BUSCO job for each
+for LOC in "${TXOMES[@]}"; do
+  sbatch "$BUSCO" "$LOC" "$LOC"/Trinity.fasta
+done

05-transcriptomes/isoseq-01-consensus.sh

+#!/usr/bin/env bash
+
+#SBATCH --job-name=pb-consensus
+#SBATCH --ntasks=1
+#SBATCH --cpus-per-task=64
+#SBATCH --mem=5G
+#SBATCH --time=5:00:00
+#SBATCH --mail-type=ALL
+#SBATCH --mail-user=nikolaos.papadopoulos@univie.ac.at
+#SBATCH --output=/lisc/user/papadopoulos/log/pycno-isoseq-ccs-%j.out
+#SBATCH --error=/lisc/user/papadopoulos/log/pycno-isoseq-ccs-%j.err
+
+module load conda
+conda activate pbccs-6.4.0
+
+PACBIO="/lisc/scratch/zoology/pycnogonum/raw/r64046_20240215_071715_C02/"
+SUBREADS=$PACBIO/m64046_240218_003256.subreads.bam
+CCS=$PACBIO/ccs.bam
+
+cd "$TMPDIR" || exit
+
+# generate CCS reads
+# use the MIMALLOC env proposed by the pbccs documentation
+MIMALLOC_PAGE_RESET=0 MIMALLOC_LARGE_OS_PAGES=1 ccs "$SUBREADS" "$CCS" --num-threads 64 --log-level INFO --report-file "$CCS".report.txt
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05-transcriptomes/isoseq-02-lima.sh

+#!/usr/bin/env bash
+
+#SBATCH --job-name=pb-demux
+#SBATCH --ntasks=1
+#SBATCH --cpus-per-task=32
+#SBATCH --mem=500M
+#SBATCH --time=5:00:00
+#SBATCH --mail-type=ALL
+#SBATCH --mail-user=nikolaos.papadopoulos@univie.ac.at
+#SBATCH --output=/lisc/user/papadopoulos/log/pycno-isoseq-demux-%j.out
+#SBATCH --error=/lisc/user/papadopoulos/log/pycno-isoseq-demux-%j.err
+
+module load conda
+conda activate lima-2.9.0
+
+PACBIO="/lisc/scratch/zoology/pycnogonum/raw/r64046_20240215_071715_C02/"
+CCS=$PACBIO/ccs.bam
+BARCODES=/lisc/user/papadopoulos/repos/pycno-seq/dev_transcriptomes/barcodes.fasta
+DEMUX=$PACBIO/fl.bam
+
+cd "$TMPDIR" || exit
+
+# demultiplex CCS reads with lima
+lima "$CCS" "$BARCODES" "$DEMUX" --isoseq --peek-guess --num-threads 32 --log-level INFO --log-file "$DEMUX".report.txt
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05-transcriptomes/isoseq-03-refine.sh

+#!/usr/bin/env bash
+
+#SBATCH --job-name=pb-refine
+#SBATCH --ntasks=1
+#SBATCH --cpus-per-task=32
+#SBATCH --mem=1G
+#SBATCH --time=10:00
+#SBATCH --mail-type=ALL
+#SBATCH --mail-user=nikolaos.papadopoulos@univie.ac.at
+#SBATCH --output=/lisc/user/papadopoulos/log/pycno-isoseq-refine-%j.out
+#SBATCH --error=/lisc/user/papadopoulos/log/pycno-isoseq-refine-%j.err
+
+module load conda
+conda activate isoseq3-4.0.0
+
+PACBIO="/lisc/scratch/zoology/pycnogonum/raw/r64046_20240215_071715_C02/processed"
+BARCODES=/lisc/user/papadopoulos/repos/pycno-seq/dev_transcriptomes/barcodes.fasta
+
+cd "$TMPDIR" || exit
+
+DEMUX=$PACBIO/fl.thylaeodus_NEB_5p--NEB_Clontech_3p.bam
+FLNC=$PACBIO/flnc.thylaeodus.bam
+isoseq refine "$DEMUX" "$BARCODES" "$FLNC" --num-threads 32 --log-level INFO --log-file "$FLNC".report.txt --require-polya
+
+DEMUX=$PACBIO/fl.scutopus_NEB_5p--NEB_Clontech_3p.bam
+FLNC=$PACBIO/flnc.scutopus.bam
+isoseq refine "$DEMUX" "$BARCODES" "$FLNC" --num-threads 32 --log-level INFO --log-file "$FLNC".report.txt --require-polya
+
+DEMUX=$PACBIO/fl.pycnogonum_NEB_5p--NEB_Clontech_3p.bam
+FLNC=$PACBIO/flnc.pycnogonum.bam
+isoseq refine "$DEMUX" "$BARCODES" "$FLNC" --num-threads 32 --log-level INFO --log-file "$FLNC".report.txt --require-polya
+
+# DEMUX=$PACBIO/fl.pycnogonum_NEB_5p--NEB_Clontech_3p.bam
+# FLNC=$PACBIO/flnc.pycnogonum-nopolyA.bam
+# isoseq refine "$DEMUX" "$BARCODES" "$FLNC" --num-threads 32 --log-level INFO --log-file "$FLNC".report.txt
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05-transcriptomes/isoseq-04-cluster.sh

+#!/usr/bin/env bash
+
+#SBATCH --job-name=pb-cluster
+#SBATCH --ntasks=1
+#SBATCH --cpus-per-task=32
+#SBATCH --mem=20G
+#SBATCH --time=60:00
+#SBATCH --mail-type=ALL
+#SBATCH --mail-user=nikolaos.papadopoulos@univie.ac.at
+#SBATCH --output=/lisc/user/papadopoulos/log/pycno-isoseq-cluster-%j.out
+#SBATCH --error=/lisc/user/papadopoulos/log/pycno-isoseq-cluster-%j.err
+
+module load conda
+# conda activate isoseq3-4.0.0
+
+PACBIO="/lisc/scratch/zoology/pycnogonum/raw/r64046_20240215_071715_C02/processed"
+
+cd "$TMPDIR" || exit
+
+FLNC=$PACBIO/flnc.thylaeodus.bam
+TRANSCRIPTS=$PACBIO/clustered.thylaeodus.bam
+isoseq cluster2 "$FLNC" "$TRANSCRIPTS" --num-threads 32 --log-level INFO
+
+FLNC=$PACBIO/flnc.scutopus.bam
+TRANSCRIPTS=$PACBIO/clustered.scutopus.bam
+isoseq cluster2 "$FLNC" "$TRANSCRIPTS" --num-threads 32 --log-level INFO
+
+FLNC=$PACBIO/flnc.pycnogonum.bam
+TRANSCRIPTS=$PACBIO/clustered.pycnogonum.bam
+isoseq cluster2 "$FLNC" "$TRANSCRIPTS" --num-threads 32 --log-level INFO
+
+
+# now convert the clustered BAM files to FASTA
+conda activate pacbio
+
+TRANSCRIPTS=$PACBIO/clustered.thylaeodus.bam
+bam2fasta -o $PACBIO/thylaeodus_isoseq --num-threads 32 "$TRANSCRIPTS"
+TRANSCRIPTS=$PACBIO/clustered.scutopus.bam
+bam2fasta -o $PACBIO/scutopus_isoseq --num-threads 32 "$TRANSCRIPTS"
+TRANSCRIPTS=$PACBIO/clustered.pycnogonum.bam
+bam2fasta -o $PACBIO/pycnogonum_isoseq --num-threads 32 "$TRANSCRIPTS"
\ No newline at end of file

05-transcriptomes/isoseq-05-align.sh

+#!/usr/bin/env bash
+
+#SBATCH --job-name=pb-align
+#SBATCH --ntasks=1
+#SBATCH --cpus-per-task=5
+#SBATCH --mem=10G
+#SBATCH --time=10:00
+#SBATCH --mail-type=ALL
+#SBATCH --mail-user=nikolaos.papadopoulos@univie.ac.at
+#SBATCH --output=/lisc/user/papadopoulos/log/pycno-isoseq-align-%j.out
+#SBATCH --error=/lisc/user/papadopoulos/log/pycno-isoseq-align-%j.err
+
+module load conda
+
+# input
+PACBIO="/lisc/scratch/zoology/pycnogonum/raw/r64046_20240215_071715_C02/processed"
+DRAFT="/lisc/scratch/zoology/pycnogonum/genome/draft/draft_softmasked.fasta"
+FLNC=$PACBIO/flnc.pycnogonum.bam
+TRANSCRIPTS=$PACBIO/clustered.pycnogonum.bam
+
+# output
+MAPPED="/lisc/scratch/zoology/pycnogonum/transcriptome/isoseq/mapped.bam"
+GFF="/lisc/scratch/zoology/pycnogonum/transcriptome/isoseq/collapsed.gff"
+
+cd "$TMPDIR" || exit
+
+conda activate pacbio
+pbmm2 align --preset ISOSEQ --sort -j 16 -J 4 -m 1200M --log-level INFO $TRANSCRIPTS $DRAFT $MAPPED
+conda deactivate
+
+conda activate isoseq3-4.0.0
+isoseq collapse --do-not-collapse-extra-5exons --log-level INFO -j 32 $MAPPED $FLNC $GFF
+conda deactivate
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