cDNA_Cupcake

Last Updated: 08.23.2022

MAJOR UPDATE (08/23/2022)!!! With many of the core Cupcake functions now moving into official isoseq software, including support for bulk and single-cell Iso-Seq data, I've removed a good chunk of outdated scripts and will no longer be addressing bugs for which there is already a isoseq equivalent (ex: isoseq3 collapse is now the preferred tool over collapse_isoforms_by_sam.py). Use the remaining code at your own risk and please work to transition to official isoseq software. I'm gradually updating the wiki through end of 2022 to show the equivalent scripts in the official v Cupcake version. Starting 2023 I will begin to remove the redundant Cupcake scripts altogether (though of course you can still access them via older tagged versions in this repo).

I foresee that I will continue to support standalone scripts such as those in sequence/ subdir, most of which require no installation and have no existing alternatives.

Current version: 29.0.0

Python Requirements

• Python >= 3.7
• Biopython

If you are running post Iso-Seq analysis you will need more install requirements. See wiki for more details.

How to use this repository

Since most of the scripts are independent (do not depend on each other), you can either clone the whole directory, or, if you are only interested in a specific script, just download that specific script to your local drive.

You can clone the GitHub repository, then add the GitHub repo path to your $PATH variable. The scripts are organized into different sub-directories (ex: sequence/, rarefaction/ etc) so you will have to add them individually.

git clone https://github.com/Magdoll/cDNA_Cupcake.git
export PATH=$PATH:<path_to_Cupcake>/sequence/
export PATH=$PATH:<path_to_Cupcake>/rarefaction/

For any issues or bugs, please report to Issues.

Documentation

Please see wiki for the latest maintained list of scripts.

A brief list of currently listed scripts are:

Annotation and Rarefaction

• make_file_for_subsampling_from_collapsed.py: Prepare file for running subsampling (rarefaction curve).
• subsample.py and subsample_with_category.py: Running subsamping. Results can be plotted with Excel graphics and R, etc.

Sequence Manipulation

• get_seq_stats.py: Summarize length distribution of a FASTA/FASTQ file.
• rev_comp.py: Reverse complement a sequence from command line.
• fa2fq.py and fq2fa.py: Convert between FASTA and FASTQ format.
• get_seqs_from_list.py: extract list of sequences given a fasta file and a list of IDs.
• err_correct_w_genome.py: generate fasta sequences given genom
• sam_to_bam.py: quick script to run SAM to BAM conversion. Assumes samtools is installed.
• sam_to_gff3.py: use BCBio and BioPython to convert SAM file into GFF3 format.
• group_ORF_sequences.py: group identical ORF sequences from different isoforms.

Cupcake ToFU: supporting scripts for Iso Seq after clustering step

• collapse_isoforms_by_sam.py: Collapse HQ isoform results to unique isoforms (based on genome alignment).
• get_abundance_post_collapse.py: Obtain count information post collapse to unique isoforms.
• filter_by_count.py: Filter collapse result by FL count information.
• filter_away_subset.py: Filter away 5' degraded isoforms.
• simple_stats_post_collapse.py: Generating simple stats file to plot in R later.
• chain_samples.py: Chaining together multiple samples.
• fusion_finder.py: Finding fusion genes.
• fusion_collate_info.py: Collate fusion information after running SQANTI(3).
• color_bed12_post_sqanti.py: Color BED12 files using FL counts after running SQANTI(3).

Version Changes

2022.08.23 updated to v29.0.0. Deprecation of modules that are no longer in use or replaced by official isoseq software.

2021.09.15 updated to v28.0.0. Fixed over-merging of isoforms in collapse_isoforms_by_sam.py and fusion_finder.py based on PR171

2021.08.11 updated to v27.0.0. adding single cell grouping to phasing support.

2021.08.06 updated to v26.2.0. added phasing/utils/tag_bam_post_phasing.py for tagging BAMs post IsoPhase. Also, demux_isoseq_with_genome.py now accepts PBfusion ID format and outputs warning for missing read info instead of failing. (do not use v26.0.0 or v26.1.0; bug in the ID matching)

2021.05.01 updated to v25.2.0. collapse_isoforms_by_sam.py now accepts bam input and also multiple CPU use.

2021.04.13 updated to v24.3.0 typo fixed in cupcake.io.BioReaders.py

2021.04.10 updated to v24.2.0 get_abundance_post_collapse.py now accepts all forms of cluster ID

2021.03.12 updated to v23.0.0. using pval to reduce non-SNP-potential positions in IsoPhase.

2021.03.09 updated to v22.0.0. fusion_collate_info.py fixed to use proper GTF for gene name to ENSG ID mapping.

2021.03.08 updated to v21.0.0. Added mag_phaser.py for metagenomics phasing.

2021.01.18 updated to v19.0.0. IsoPhase release (for redwood Iso-Seq) and updated fusion_collate_info.py

2020.12.15 updated to v18.1.0. fixed file splitting bug in chain_samples.py.

2020.12.14 updated to v18.0.0. added simple_stats_post_collapse.py.

2020.12.03 updated to v17.1.0. added color_bed12_post_sqanti.py

2020.11.20 updated to v16.0.0. collapse_isoforms_by_sam.py has --gen_mol_count option to output .abundance.txt output (for FLNC or UMI-based isoseq3 dedup mappings)

2020.11.12 updated to v15.1.0. fusion_collate_info.py can now work without .abundance.txt

2020.11.12 updated to v15.0.0. summarized VCF file from collect_all_vcf.py in IsoPhase now will output a SAMPLE column.

Click to see older version logs

2020.10.17 updated to v14.2.0. added `fusion_collate_info.py` which was missing in v14.0.0 causing install failure.

2020.10.14 updated to v14.0.0. `fusion_finder.py` now outputs fusions in transcription order.

2020.09.23 updated to v13.0.0. adding support for `isoseq3 dedup`

2020.09.15 updated to v12.5.0. fixed bug for `chain_samples.py` where chromosomes are numeric (ex: 12 instead of chr12).

2020.08.12 updated to v12.4.0. fixed bug for `chain_samples.py` for a chunk sizing bug.

2020.08.05 updated to v12.3.0. fixed bug for `demux_isoseq_with_genome.py` to work with mapped fasta output in SL9.

2020.07.15 updated to v12.2.0. fixed bug for `clip_out_UMI_cellBC.py` for MS characters - switched to unix dialect.

2020.05.28 updated to v12.1.0. fixed bug for `clip_out_UMI_cellBC.py` handling cigar strings.

2020.01.31 updated to v10.0.1. fixed typo in `collapse_isoforms_by_sam.py`

2020.01.27 updated to v10.0.0. `chain_samples.py` now supported multithreading via `--cpus` option, also fixed chain bugs related to 3' differences.

2019.12.30 updated to v9.2.0. fixed support for running `run_preCluster.py` that is used by Cogent.

2019.12.18 updated to v9.1.1. changed `bcbiogff` dependency to `bcbio-gff` in `setup.py`.

2019.12.18 updated to v9.1.0. added fasta support for `filter_away_subset.py` and `filter_by_count.py`.

2019.12.09 updated to v9.0.3. fixed geneid display issue with PB.X.Y in GFF output for collapse.

2019.10.02 updated to v9.0.2. updated collapse script series parameters to fit with isoseq3 output.

2019.10.02 updated to v9.0.1. bug fix on fasta output in `dedup_FLNC_per_cluster.py`. removed pbcore dependency so Py3 fully usable for single cell scripts!

2019.09.25 updated to v8.7.0. `clip_out_UMI_cellBC.py` supports unusual 10X 5' end single cell schema.


2019.09.24 updated to v8.6. `cupcake.io.GFF.py` now supports `gene_id` write out.

2019.09.16 updated to v8.5. fixed `collapse_isoforms_by_sam.py` incorrect behavior in fuzzy chain

2019.08.20 updated to v8.4. `run_phaser.py` dependncy is pyvcf, not bio-vcf.

2019.08.19 updated to v8.3. removed bug testing code in cupcake preclustering.

2019.07.26 updated to v8.2. `subsample.py` now uses `min_fl_count` cutoff in count setup. also added `subsample_with_category.py`

2019.07.25 updated to v8.1. `sam_to_gff3.py` modified to work with SQANTI2 (v3.3+) changes

2019.07.02 updated to v8.0. `cupcake.io.GFF.GTF` now can handle missing transcript_name field

2019.06.25 updated to v7.9. fixed minor dict issue with `demux_by_barcode_groups.py`

2019.06.21 updated to v7.8. fixed `demux_by_barcode_groups.py` tab/space mixing issue.

2019.06.20 updated to v7.7. fixed chromosome output error in `chain_fusion_samples.py`.

2019.06.07 updated to v7.6. changed preClustering to include "tucked" sequences

2019.06.03 updated to v7.5. added `summarize_byloci_results.py` and `collect_all_vcf.py` in phasing/ for IsoPhase.

2019.05.28 updated to v7.4. fixed `select_loci_to_phase.py` to work for short genomes.

2019.05.22 updated to v7.3. made `calc_expected_accuracy_from_fastq.py` and `filter_lq_isoforms.py` work for FLNC. Also added 0-bp exon filter for collapse script.

2019.04.30 updated to v7.2. fixed warning/bug in `coordinate_mapper.py` by use `str()` instead of `.tostring()` for Bio.Seq objects.

2019.04.30 updated to v7.1. added `group_ORF_sequences.py` for grouping ORF predictions.

2019.04.08 updated to v7.0. fixed `summarize_sample_GFF_junctions.py` for newline error.

2019.03.27 updated to v6.9. fixed `clip_out_UMI_cellBC.py` to properly handle 0-length UMIs or BCs (but not both).

2019.03.19 updated to v6.8. fixed `phasing.io.SAMMPileUpReader.py` for cov 0 returns

2019.03.14 updated to v6.7. added `sam_to_collapsed_gff.py`

2019.03.11 updated to v6.6. temp support of lazy  BED reader in BED.py

2019.02.25 updated to v6.5. fixed `filter_away_subset.py` to handle edge case where the shorter one is monoexonic.

2019.01.31 updated to v6.4. fixed junction 6-field support in `scrub_sample_GFF_junctions.py`. 

2019.01.30 updated to v6.3. fixed typo in `summarize_sample_GFF_junctions.py`.

2019.01.12 updated to v6.2. added first version of IsoPhase scripts.

2018.10.29 updated to v6.1. changed confusing param name in `chain_samples.py` to `--dun-merge-5-shorter`

2018.10.29 updated to v6.0. added `demux_by_barcode_group.txt` for creating demultiplexed GFF (and FASTX) from demux count files.

2018.10.15 updated to v5.11. `sam_to_gff3.py` updated to allow `source` param.

2018.10.12 updated to v5.10. collapse scripts further handles isoseq3 with mapping formats.

2018.08.30 updated to v5.9. have collapse script handle isoseq3 formats correctly in get_fl_from_id().

2018.08.01 updated to v5.8. (also tagged as `cupcake_v5.8`) fixed `sam_to_gff3.py` to output GFF3 correctly, also refreshed BioReaders.py in sequence/ to be up-to-date with cupcake/io version.

2018.07.16 updated to v5.7. added `sam_to_bam.py` and `sam_to_gff3.py` (requires BCBio)

2018.07.13 updated to v5.6. fixed polyA length bug in make classify report for isoseq3.

2018.06.29 updated to v5.4. collapse,fusion,abundance,demux now works with isoseq3 output. 

2018.03.29 updated to v5.3. Update to work with pitchfork SA5.1

2018.03.12 updated to v5.2. Fixed over-collapsing genes in collapse script. Now processing strands separately in correct manner.

2017.11.06 updated to v4.1. pCS merge incorrect in `chain_samples.py`. Fixed.

2017.10.31 updated to v4.0. pCS merge incorrect in `run_preCluster.py`. Fixed.

2017.10.10 updated to v3.9. Merged pCS branch (`--dun_use_partial`) and cdunn's random seed.

2017.09.25 updated to v3.7. Fixed minor printing error in `scrubbed.group.txt` for `scrub_sample_GFF_junctions.py`.