RASLseq FASTQ reads to RASLprobe counts.
RASL-seq is a powerful and inexpensive method to assess gene expression without the need for RNA isolation1. We recently published a modified protocol using the RNA ligase Rnl2 which demonstrated dramatically increased ligation efficiency2. This python package offers an alignment method leveraging BLASTn, pandas, py-editdist, and NumPy. Optimizations will follow in the near future.
python /path/to/RASLseqAnalysis.py [required args -f -s -p -w -d -b -o] [optional args -P]
-f : absolute path to fastq file (accepts gzip files)
-p : absolute path to probes file
-w : absolute path to annotations file
-d : absolute path to write directory for blast database
-b : absolute path to blast bin directory
-o : absolute path of output file
-s : specifies sequencer id in fastq index line, e.g. @HISEQ
-P : verbose printing
example command:
python /path/to/RASLseqAnalysis.py -f /path/to/your.fastq.gz -s @HISEQ -p /paht/to/RASL.probes -w /path/to/annotations.bc -d /path/to/blastdb/write_dir/ -b /path/to/blast/ncbi-blast-2.2.26+/bin/ -P -o /path/to/output.txtexample data: can be found in the data directory
NOTE:RASLseqAnalysis_NAR.py is provided for transparency and requires manual parameter settings to run
FASTQ: standard FASTQ format (optionally gzipped)
X.probes: tab-separated file describing the RASLseq Probes with the following columns and column headers
AcceptorProbeSequence
DonorProbeSequence
AcceptorAdaptorSequence
DonorAdaptorSequence
ProbeName
Please see example file in data/ directory
X.bc: tab-separated file describing each well in the experiment with the following columns and column headers
REQUIRED:
plate_bc
well_bc
OPTIONAL: additional columns with well metadata, column headers are user defined, e.g. drug_concentration
Please see example file in data/ directory
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BLASTn
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pandas
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py-editdist
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NumPy
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H. Li, J. Qiu, X.-D. Fu, RASL-seq for massive parallel and quantitative analysis of gene expression, Curr. Protocol. Mol. Biol., 98 (2012), pp. 4.13.1–4.13.9
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Larman HB, Scott ER, Wogan M, Oliveira G, Torkamani A, Schultz PG, Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay, Nucleic Acids Res. 2014;42(14):9146-57