Genetic Code Prediction and Annotation in Uncultivated Ciliates from Marine Environments

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Genetic code prediction from karyorelict and heterotrich ciliates

Among the ciliates, there is an unusually diverse number of genetic codes used by different species, compared with other groups of eukaryotes. The karyorelicts and heterotrichs have some of the most unusual types of codes, where stop codons are potentially ambiguous and can also code for amino acids.

Aims:

Assemble single cell transcriptome RNAseq data from uncultivated ciliates
Assemble whole-genome-amplification DNAseq data from uncultivated ciliates
Predict genetic codes from above assembles
Annotate genes from above assemblies, identify clear examples of ambiguous stop genetic codes

See our preprint (doi:10.1101/2022.04.12.488043) for more information.

Data

Single-cell transcriptome RNAseq from uncultivated marine ciliates, collected from Roscoff, France
Published datasets downloaded from SRA

Data deposition

Primary read data from this study: ENA PRJEB50648
Snakemake pipelines:

Code Snippets

shell:
    "phyloFlash.pl -lib {wildcards.lib}_rnaseq_maponly_{wildcards.readlim} -readlength 150 -readlimit {wildcards.readlim} -skip_spades -read1 {input.fwd} -read2 {input.rev} -CPUs {threads} -html -treemap -log -poscov -zip -dbhome {params.db} 2> {log};"
    "mv {wildcards.lib}_rnaseq_maponly_{wildcards.readlim}.phyloFlash* qc/phyloFlash/;"

SnakeMake From line 80 of main/Snakefile

shell:
    "phyloFlash.pl -lib {wildcards.lib}_dnaseq -readlength 150 -read1 {input.fwd} -read2 {input.rev} -CPUs {threads} -almosteverything -dbhome {params.db} 2> {log};"
    "mv {wildcards.lib}_dnaseq.phyloFlash* qc/phyloFlash/;"

SnakeMake From line 112 of main/Snakefile

shell:
    "bbduk.sh -Xmx10g threads={threads} ref=resources/adapters.fa,resources/phix174_ill.ref.fa.gz in={input.fwd} in2={input.rev} ktrim=r qtrim=rl trimq=24 minlength=25 out={output.fwd} out2={output.rev} 2> {log}"

SnakeMake BBMap From line 126 of main/Snakefile

shell:
    "bbduk.sh -Xmx10g threads={threads} ref=resources/adapters.fa,resources/phix174_ill.ref.fa.gz in={input.fwd} in2={input.rev} ktrim=r qtrim=rl trimq=24 minlength=25 out={output.fwd} out2={output.rev} 2> {log}"

SnakeMake BBMap From line 139 of main/Snakefile

shell:
    "Trinity --seqType fq --max_memory 64G --bflyHeapSpaceMax 40G --CPU {threads} --full_cleanup --left {input.fwd} --right {input.rev} --output {params.prefix} &> {log};"

SnakeMake seqtk-trinity From line 153 of main/Snakefile

shell:
    "cat {input.assem} | parallel --gnu -j {threads} --recstart '>' -N 100 --pipe blastx -query - -db {params.db_prefix} -evalue 1e-20 -max_target_seqs 1 -outfmt 6 > {output.blastx};"
    "analyze_blastPlus_topHit_coverage.pl {output.blastx} {input.assem} {input.db} &> {log};"

SnakeMake From line 171 of main/Snakefile

shell:
    "bbduk.sh -Xmx10g threads={threads} ref=resources/adapters.fa,resources/phix174_ill.ref.fa.gz in={input} ktrim=r qtrim=rl trimq=24 minlength=25 out={output} 2> {log}"

SnakeMake BBMap From line 184 of main/Snakefile

shell:
    "Trinity --seqType fq --max_memory 64G --bflyHeapSpaceMax 40G --CPU {threads} --full_cleanup --single {input} --output {params.prefix} &> {log};"