Snakemake pipeline for de novo transcriptome assembly with 454 reads

public public 1yr ago 0 bookmarks
View Workflow
Report
smsk_454
smsk_454
View Workflow

1. Description

This is a workflow to assemble RNA reads from 454 into a transcriptome. The procedure is as follows:

  1. Quality control

    1. Base calling with PyroBayes (if .sff files are provided)

    2. Quality, length and adaptor trimming with SnoWhite

  2. Assembly

    With gsAssembler from Roche's Data Analysis tools ( newbler )

2. First steps

  1. Clone the repo 

    git clone https://github.com/jlanga/smsk_454.git # Clone
cd smsk_454

  2. Activate the environment ( deactivate to deactivate): 

    source bin/activate

  3. Install software and packages via pip and homebrew (edit whatever is necessary): 

    bash bin/install/brew.sh
bash bin/install/from_brew.sh
bash bin/install/from_pip3.sh
bash bin/install/from_tarball.sh

  4. Additional requirements

    Pyrobayes is an accurate base caller for 454 datasets. It used to be available through free registration at here . It used to be a file called pyrobayes.unified_release_64bit.tar.gz . If you are able to get it, do the following 

    mkdir -p src/
pushd src/
cp /path/to/tarball.tar.gz .
tar xvf pyrobayes.unified_release_64bit.tar.gz
popd
cp src/UnifiedRelease/bin/PyroBayes bin/

    The same applies to get gsAssembler . You should go to Roche and ask for a copy (it is free but requires registration). You should get a file called DataAnalysis_2.9_All_20130530_1559.tgz . If you are connecting through ssh, use the -X option to allow graphic interfaces ( ssh server -X ).

    From here, 

    mkdir -p src/
pushd src/
cp /path/to/DataAnalysis_2.9_All_20130530_1559.tgz .
tar xvf DataAnalysis_2.9_All_20130530_1559.tgz
pushd DataAnalysis_2.9_All/
bash setup.sh

    And a window will pop up. Select "local installation" and choose as installation path the src/ directory of this project (in my case /home/jlanga/pipelines/smsk_454/src/454/ )

  5. Download sample data from the European Nucleotide Archive (ENA; two sff files and two fastq files): 

    bash bin/download_test_data.sh

  6. Execute the pipeline (should take up to 10 minutes): 

    snakemake -j 24

3. File organization

The hierarchy of the folder is the one described in A Quick Guide to Organizing Computational Biology Projects : 

smsk
├── .linuxbrew: brew files
├── bin: scripts,binaries and snakemeake related files.
├── data: raw data, hopefully links to backuped data.
├── doc: logs.
├── README.md
├── results: processed data, reports, etc.
└── src: additional source code, tarballs, etc.

Bibliography and resources

Code Snippets

 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
from Bio import SeqIO
from Bio.SeqIO.QualityIO import PairedFastaQualIterator
import sys

def convert(fasta_file, qual_file, fastq_file):
    """
    Combine fasta_file and qual_file into fastq_file in PHRED33 qual format.
    """
    SeqIO.write(
        (record for record in PairedFastaQualIterator(fasta_file, qual_file)),
        fastq_file,
        "fastq"
    )


if __name__ == "__main__":

    if len(sys.argv) != 3:
        sys.exit("ERROR! Provide a fasta and a qual file. Output will be spitted through stdout")

    fasta = open(sys.argv[1], "r")
    qual  = open(sys.argv[2], "r")
    fastq = sys.stdout

    convert(fasta, qual, fastq)
Python Biopython From line 3 of bin/fasta_qual_to_fastq.py 
30
31
32
33
34
35
36
37
38
39
40
41
42
43
shell:
    "./src/454/bin/runAssembly "
        "-cdna "
        "-cpu {threads} "
        "-minlen {params.minimum_sequence_length} "
        "-mi {params.minimum_overlap_identity} "
        "-ml {params.minimum_overlap_length} "
        "-o {params.out_dir} "
        "-vs {input.adaptors} "
        "-vt {input.adaptors} "
        "{params.additional_params} "
        "{input.sff} "
        "{input.fastq} "
    "2> {log} 1>&2"
SnakeMake From line 30 of snakefiles/assembly 
5
6
shell:
    "rm -r results/"
SnakeMake From line 5 of snakefiles/clean 
14
shell: "rm -r {raw_dir}"
SnakeMake From line 14 of snakefiles/clean 
22
shell: "rm -r {qc_dir}"
SnakeMake From line 22 of snakefiles/clean 
30
shell: "rm -r {assembly_dir}"
SnakeMake From line 30 of snakefiles/clean 
38
shell: "rm -r {report_dir}"
SnakeMake From line 38 of snakefiles/clean 
15
16
17
18
19
20
21
shell:
    "pigz "
        "--decompress "
        "--stdout "
        "{input.fq_gz} "
    "> {output.fq} "
    "2> {log}"
SnakeMake From line 15 of snakefiles/qc_fastq 
 68
 69
 70
 71
 72
 73
 74
 75
 76
 77
 78
 79
 80
 81
 82
 83
 84
 85
 86
 87
 88
 89
 90
 91
 92
 93
 94
 95
 96
 97
 98
 99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
shell:
    "tar "
        "xvf "
        "{params.tarball} "
        "-C . "
    "2> {log} 1>&2 ; "
    "mv SnoWhite_2.0.3_release/* . ; "
    "yes | perl snowhite_2.0.3.pl "
        "-f {input.fastq} "
        "-o {params.out_folder} "
        "-v {input.adaptors} "
        "-s {input.adaptors} "
        "-m {params.minimum_sequence_length} "
        "-g {params.delete_temporary_files} "
        "-R {params.output_fastq} "
        "`#-B {params.split_in_subfiles}` "
        "`#-j {params.barcodes_in_end}` "
        "`#-z {params.number_of_mismatches}` "
        "-Q {params.minimum_quality} "
        "`#-E {input.adaptors}` "
        "`#-c {params.bases_to_clip}` "
        "-D {params.execute_tagdust} "
        "-d {params.false_discovery_rate} "
        "-L {params.execute_seqclean} "
        "-p {threads} "
        "-Y {params.execute_poly_trimming} "
        "-l {params.poly_tail_min_repeat_length} "
        "-a {params.poly_a_ends} "
        "-t {params.poly_t_ends} "
        "-b {params.poly_tail_terminal_bases_cap} "
        "-r {params.poly_tail_min_repeat_length} "
        "-i {params.internal_poly_minimum_length} "
        "-k {params.keep_longer_end} "
        "-n {params.ns_as_a_or_t} "
        "-w {params.allow_wobble} "
    "2>> {log} 1>&2 || true ; " #Snowhite returns exit status different from 0 even after success
    "mv "
        "out/FinalOutput/out.clean "
        "{output.fastq} "
    "2>> {log} 1>&2 ; "
    "pigz "
        "--best "
        "--stdout "
        "{output.fastq} "
    "> {output.fastq_gz} "
    "2>> {log}"
SnakeMake From line 68 of snakefiles/qc_fastq 
152
153
154
155
156
157
158
159
shell:
    "trimmomatic SE "
        "-threads {threads} "
        "-phred33 "
        "<(pigz -dc {input.fq_gz}) "
        ">(cut -f 1 -d \" \" | pigz -9 > {output.fq_gz}) "
        "{params.trimmomatic_params} "
    "2> {log}"
SnakeMake Trimmomatic From line 152 of snakefiles/qc_fastq 
177
178
179
shell:
    "pigz "
        "--decompress "
SnakeMake From line 177 of snakefiles/qc_fastq 
18
19
20
21
22
shell:
    "./bin/PyroBayes "
        "--sffFile {input.sff} "
        "--outStub {params.stub} "
    "2> {log} 1>&2"
SnakeMake From line 18 of snakefiles/qc_sff 
41
42
43
44
45
46
shell:
    "( python3 bin/fasta_qual_to_fastq.py "
        "{input.fasta} "
        "{input.qual} | "
    "pigz --best > {output.fastq_gz} ) "
    "2> {log} 2>&1"
SnakeMake From line 41 of snakefiles/qc_sff 
64
65
66
67
68
69
70
shell:
    "pigz "
        "--decompress "
        "--stdout "
        "{input.fq_gz} "
    "> {output.fq} "
    "2> {log}"
SnakeMake From line 64 of snakefiles/qc_sff 
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
shell:
    "tar "
        "xvf "
        "{params.tarball} "
        "-C . "
    "2> {log} 1>&2 ; "
    "mv SnoWhite_2.0.3_release/* . ; "
    "yes | perl snowhite_2.0.3.pl "
        "-f {input.fastq} "
        "-o {params.out_folder} "
        "-v {input.adaptors} "
        "-s {input.adaptors} "
        "-m {params.minimum_sequence_length} "
        "-g {params.delete_temporary_files} "
        "-R {params.output_fastq} "
        "`#-B {params.split_in_subfiles}` "
        "`#-j {params.barcodes_in_end}` "
        "`#-z {params.number_of_mismatches}` "
        "-Q {params.minimum_quality} "
        "`#-E {input.adaptors}` "
        "`#-c {params.bases_to_clip}` "
        "-D {params.execute_tagdust} "
        "-d {params.false_discovery_rate} "
        "-L {params.execute_seqclean} "
        "-p {threads} "
        "-Y {params.execute_poly_trimming} "
        "-l {params.poly_tail_min_repeat_length} "
        "-a {params.poly_a_ends} "
        "-t {params.poly_t_ends} "
        "-b {params.poly_tail_terminal_bases_cap} "
        "-r {params.poly_tail_min_repeat_length} "
        "-i {params.internal_poly_minimum_length} "
        "-k {params.keep_longer_end} "
        "-n {params.ns_as_a_or_t} "
        "-w {params.allow_wobble} "
    "2>> {log} 1>&2 || true ; " #Snowhite returns exit status different from 0 even after success
    "mv "
        "out/FinalOutput/out.clean "
        "{output.fastq} "
    "2>> {log} 1>&2 ; "
    "pigz "
        "--best "
        "--stdout "
        "{output.fastq} "
    "> {output.fastq_gz} "
    "2>> {log}"
SnakeMake From line 117 of snakefiles/qc_sff 
207
208
209
210
211
212
213
214
215
shell:
    "trimmomatic SE "
        "-threads {threads} "
        "-phred33 "
        "<(pigz -dc {input.fq_gz}) "
        ">(cut -f 1 -d \" \" | pigz -9 > {output.fq_gz}) "
        "{params.trimmomatic_params} "
    "2> {log} ; "
    "sleep 5s"
SnakeMake Trimmomatic From line 207 of snakefiles/qc_sff 
233
234
235
236
shell:
    "sleep 5s ; "
    "pigz "
        "--decompress "
SnakeMake From line 233 of snakefiles/qc_sff 
13
14
15
16
17
shell:
    "ln --symbolic "
        "$(readlink --canonicalize {input.sff} "
        "{output.sff}) "
    "2> {log}"
SnakeMake From line 13 of snakefiles/raw 
33
34
35
36
37
shell:
    "ln --symbolic "
        "$(readlink --canonicalize {input.fastq} "
        "{output.fastq}) "
    "2> {log}"
SnakeMake From line 33 of snakefiles/raw 
55
56
57
58
59
60
61
62
shell:
    "(python3 bin/fasta_qual_to_fastq.py "
        "<(./src/454/bin/sffinfo -s {input.sff}) "
        "<(./src/454/bin/sffinfo -q {input.sff}) | "
    "pigz "
        "--best "
    "> {output.fq_gz} ) "
    "2> {log}"
SnakeMake From line 55 of snakefiles/raw 
80
81
82
83
84
shell:
    "ln --symbolic "
        "$(readlink --canonicalize {input.fa}) "
        "{output.fa} "
    "2> {log}"
SnakeMake From line 80 of snakefiles/raw 
18
19
20
21
22
23
shell:
    "fastqc "
        "--nogroup "
        "--outdir {params.outdir} "
        "{input.fastq} "
    "> {log} 2>&1"
SnakeMake FastQC From line 18 of snakefiles/report 
44
45
46
47
48
49
shell:
    "fastqc "
        "--nogroup "
        "--outdir {params.outdir} "
        "{input.fastq} "
    "> {log} 2>&1"
SnakeMake FastQC From line 44 of snakefiles/report 
70
71
72
73
74
75
shell:
    "fastqc "
        "--nogroup "
        "--outdir {params.outdir} "
        "{input.fastq} "
    "> {log} 2>&1"
SnakeMake FastQC From line 70 of snakefiles/report 
 96
 97
 98
 99
100
101
shell:
    "fastqc "
        "--nogroup "
        "--outdir {params.outdir} "
        "{input.fastq} "
    "> {log} 2>&1"
SnakeMake FastQC From line 96 of snakefiles/report 
128
129
130
131
132
133
134
135
136
137
138
139
140
141
shell:
    "echo {input} | "
    "tr -s \" \" | "
    "tr \" \" \"\\n\" "
    "> {params.fofn} 2> {log} ; "
    "multiqc "
        "--fullnames "
        "--title {params.title} "
        "--filename {output.html} "
        "--template {params.template} "
        "--file-list {params.fofn} "
        "--data-format {params.data_format} "
        "{params.additional_params} "
    "2>> {log} 1>&2 ; "
SnakeMake MultiQC From line 128 of snakefiles/report 
164
165
166
167
168
169
shell:
    "fastqc "
        "--nogroup "
        "--outdir {params.outdir} "
        "{input.fastq} "
    "> {log} 2>&1"
SnakeMake FastQC From line 164 of snakefiles/report 
190
191
192
193
194
195
shell:
    "fastqc "
        "--nogroup "
        "--outdir {params.outdir} "
        "{input.fastq} "
    "> {log} 2>&1"
SnakeMake FastQC From line 190 of snakefiles/report 
216
217
218
219
220
221
shell:
    "fastqc "
        "--nogroup "
        "--outdir {params.outdir} "
        "{input.fastq} "
    "> {log} 2>&1"
SnakeMake FastQC From line 216 of snakefiles/report 
247
248
249
250
251
252
253
254
255
256
257
258
259
260
shell:
    "echo {input} | "
    "tr -s \" \" | "
    "tr \" \" \"\\n\" "
    "> {params.fofn} 2> {log} ; "
    "multiqc "
        "--fullnames "
        "--title {params.title} "
        "--filename {output.html} "
        "--template {params.template} "
        "--file-list {params.fofn} "
        "--data-format {params.data_format} "
        "{params.additional_params} "
    "2>> {log} 1>&2 ; "
SnakeMake MultiQC From line 247 of snakefiles/report 
311
312
313
314
315
316
317
318
319
320
321
322
323
324
shell:
    "echo {input} | "
    "tr -s \" \" | "
    "tr \" \" \"\\n\" "
    "> {params.fofn} 2> {log} ; "
    "multiqc "
        "--fullnames "
        "--title {params.title} "
        "--filename {output.html} "
        "--template {params.template} "
        "--file-list {params.fofn} "
        "--data-format {params.data_format} "
        "{params.additional_params} "
    "2>> {log} 1>&2"
SnakeMake MultiQC From line 311 of snakefiles/report 
352
353
354
355
356
357
358
359
360
361
362
363
364
365
shell:
    "echo {input} | "
    "tr -s \" \" | "
    "tr \" \" \"\\n\" "
    "> {params.fofn} 2> {log} ; "
    "multiqc "
        "--fullnames "
        "--title {params.title} "
        "--filename {output.html} "
        "--template {params.template} "
        "--file-list {params.fofn} "
        "--data-format {params.data_format} "
        "{params.additional_params} "
    "2>> {log} 1>&2"
SnakeMake MultiQC From line 352 of snakefiles/report 
389
390
391
392
393
394
395
396
397
398
399
400
401
402
shell:
    "echo {input} | "
    "tr -s \" \" | "
    "tr \" \" \"\\n\" "
    "> {params.fofn} 2> {log} ; "
    "multiqc "
        "--fullnames "
        "--title {params.title} "
        "--filename {output.html} "
        "--template {params.template} "
        "--file-list {params.fofn} "
        "--data-format {params.data_format} "
        "{params.additional_params} "
    "2>> {log} 1>&2"
SnakeMake MultiQC From line 389 of snakefiles/report 
430
431
432
433
434
435
436
437
438
439
440
441
442
443
shell:
    "echo {input} | "
    "tr -s \" \" | "
    "tr \" \" \"\\n\" "
    "> {params.fofn} 2> {log} ; "
    "multiqc "
        "--fullnames "
        "--title {params.title} "
        "--filename {output.html} "
        "--template {params.template} "
        "--file-list {params.fofn} "
        "--data-format {params.data_format} "
        "{params.additional_params} "
    "2>> {log} 1>&2"
SnakeMake MultiQC From line 430 of snakefiles/report 
471
472
473
474
475
476
477
478
479
480
481
482
483
484
shell:
    "echo {input} | "
    "tr -s \" \" | "
    "tr \" \" \"\\n\" "
    "> {params.fofn} 2> {log} ; "
    "multiqc "
        "--fullnames "
        "--title {params.title} "
        "--filename {output.html} "
        "--template {params.template} "
        "--file-list {params.fofn} "
        "--data-format {params.data_format} "
        "{params.additional_params} "
    "2>> {log} 1>&2"
SnakeMake MultiQC From line 471 of snakefiles/report
ShowHide 31 more snippets with no or duplicated tags.

Login to post a comment if you would like to share your experience with this workflow.

Do you know this workflow well? If so, you can request seller status , and start supporting this workflow.

Free

Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://github.com/jlanga/smsk_454
Name: smsk_454
Version: 1
Badge:
workflow icon

Insert copied code into your website to add a link to this workflow.

Downloaded: 0
Copyright: Public Domain
License: MIT License
  • Future updates

Related Workflows

psychip_snakemake
Show Details View Workflow
ENCODE pipeline for histone marks developed for the psychENCODE project
public

public

psychip pipeline is an improved version of the ENCODE pipeline for histone marks developed for the psychENCODE project. The o...
raw sequence reads Alignment Sequence alignment report macs2 ucsc-bedclip bedGraphToBigWig BEDTools BWA Picard SAMtools Snakemake
Free
74
ncov_2
Show Details View Workflow
Near-real time tracking of SARS-CoV-2 in Connecticut
public

public

Repository containing scripts to perform near-real time tracking of SARS-CoV-2 in Connecticut using genomic data. This pipeli...
JSON nextclade Augur Biopython FOCUS Pandas Snakemake bs4 epiweeks geopy matplotlib numpy pycountry pycountry-convert uszipcode
Free
28
cellranger-snakemake-gke
Show Details View Workflow
snakemake workflow to run cellranger on a given bucket using gke.
public

public

A Snakemake workflow for running cellranger on a given bucket using Google Kubernetes Engine. The usage of this workflow ...
macs2 ucsc-bedclip bedGraphToBigWig BEDTools BWA Picard SAMtools Snakemake
Free
74
atlas
Show Details View Workflow
ATLAS - Three commands to start analyzing your metagenome data
public

public

Metagenome-atlas is a easy-to-use metagenomic pipeline based on snakemake. It handles all steps from QC, Assembly, Binning, t...
raw sequence reads Genome assembly Annotation track checkm2 gunc prodigal snakemake-wrapper-utils MEGAHIT Atlas BBMap Biopython BioRuby Bwa-mem2 cd-hit CheckM DAS Diamond eggNOG-mapper v2 MetaBAT 2 Minimap2 MMseqs MultiQC Pandas Picard pyfastx SAMtools SemiBin Snakemake SPAdes SqueezeMeta TADpole VAMB CONCOCT ete3 gtdbtk h5py networkx numpy plotly psutil utils metagenomics
Free
175
rna-seq-star-deseq2
Show Details View Workflow
RNA-seq workflow using STAR and DESeq2
public

public

This workflow performs a differential gene expression analysis with STAR and Deseq2. The usage of this workflow is described ...
raw sequence reads Gene expression profiling ID list snakemake-wrapper-utils biomaRt PCAtools BiocParallel BWA Cutadapt DESeq2 GFFutils MultiQC Pandas SAMtools Snakemake STAR cli stringr tidyverse RNA-Seq
Free
41
dna-seq-gatk-variant-calling
Show Details View Workflow
This Snakemake pipeline implements the GATK best-practices workflow
public

public

This Snakemake pipeline implements the GATK best-practices workflow for calling small germline variants. The usage of thi...
VCF raw sequence reads Variant calling genetic variants gatk rust-bio-tools snakemake-wrapper-utils tabix BCFtools BWA FastQC MultiQC Pandas Picard SAMtools Snakemake Trimmomatic Variant Effect Predictor (VEP) common matplotlib numpy seaborn DNA
Free
57