[jaas]

Here's the config file. I can't figure out how to send it to you alone. I have changed the extension to a txt file to be able to upload it.

Thanks in advance for your help

[StevenW]

FastQ Screen Bisulfite Problem

Hi jaas,

I am one of the developers of FastQ Screen. Hopefully we can get this problem resolved quickly.

Would you be able to send me the configuration file you used when running FastQ Screen. This will help me resolve the problem.

Many thanks,

Steven

[jaas]

FastQ Screen for bisulfite samples

Hello,

I'm trying to use fastq screen for bisulfite sequencing samples. I've run the test data and that works fine. However, I get a file handle error when running my bisulfite samples:
my code:

fastq_screen --bisulfite G3.S22.fastq.gz 

Output:

Using fastq_screen v0.11.2
Defaulting to Bowtie 2 for --bisulfite mode
Reading configuration from '/data/Bismark/fastq_screen_v0.11.2/fastq_screen.conf'
Using '/usr/lib/bowtie2/bin/bowtie2' as Bowtie 2 path
Using '/data/Bismark/bismark' as Bismark path
Adding database Daphnia
Using 8 threads for searches
Option --subset set to 100000 reads
Processing G3.S22.fastq.gz
Counting sequences in G3.S22.fastq.gz
Making reduced sequence file with ratio 69:1
Searching G3.S22.fastq.gz_temp_subset.fastq against Daphnia
open: No such file or directory
[main_samview] fail to open "/data/Bismark/fastq_screen_v0.11.2/Daphnia.G3.S22.fastq.gz_temp_subset_bismark_bt2.bam" for reading.
Cannot close filehandle on '/data/Bismark/fastq_screen_v0.11.2/Daphnia.G3.S22.fastq.gz_temp_subset_bismark_bt2.bam' :  at fastq_screen line 1059. 

I do get the outputfile and mapping report of the subsample against the first database, so it seems that the mapping did work. It happens regardless of the databases I use. However when using my samples in non bisulfite mode, and mapping them against the regular genome indices, this does not happen. So I do not think my sample file is this issue. Also, I know my bismark genome build indices are fine as I used them with bismark as well.

Any ideas on what is wrong or why this is happening?

Thanks!

[StevenW]

Hi,
Thanks for your message; I am part of the team responsible for developing FastQ Screen.
We removed the --paired option from the script in a recent update as we felt it was unnecessary and was causing confusion. Mapping forward or reverse reads independently should be perfectly adequate to ascertain whether there is contamination, and will also provide the user with additional information if the forward reads are more prone generally to contamination than the reverse reads (or vice versa). Also, some users were reporting that the script was sometimes failing to detect contamination in --paired mode. For example, if the read pair did not constitute a contiguous region of DNA, or if the paired reads were separated by are large distance (such as RNA seq).
So we now recommend that you screen both read files independently.
Is there any particular reason you would have to use the –paired mode?

[turnersd]

hey, what ever happened to the --paired option? Is it still possible to screen paired end data?

[StevenW]

Excellent, so everything is fine?

PS I'm releasing a new version of the software today.

[touchsk]

Thanks Steven

I apologize for the delayed reply.

Yes, I am doing similar to what you have mentioned. I have two config files in place:
- one with just the contaminants for filtering the fastq file as you mentioned
- and one with contaminants and mammalian genomes to generate a figure using your tool that depicts the level of contamination in comparison to its real hit in a mammal (similar plot to the example on the fast_screen webpage.

This is single-end data.

The question on the single-hit was motivated because we have some contaminant-like sequences (custom to our study), that we want to quantify but not remove if they have homologs in mammalian genomes. But again using a similar approach as above, I am able to tackle that (stepwise).

Thanks for a very useful tool.

[StevenW]

Hi,
I am responsible for developing FastQ Screen.

The standard way to remove contamination is:
Run FastQ Screen (latest version) with --subset (to process the entire dataset) and --nohits. In the config file include the Bowtie1/2 indices of all the potential contaminants (human genome indices should not be included).

A FastQ file should then be produced containing all the reads that did not map to any of the contaminants.

I am wondering, why do you need to only remove the hits that are classified as 'one-hit/one-library' AND 'multiple-hits/one-library'? Also is this single-end data?

Please feel free to contact me directly to discuss this further.

Kindest regards,
Steven W

[touchsk]

Remove only 'one-hit/one-library' hits

I am trying to use FASTQ Screen to remove contaminated sequences from my data and have a question. I was looking at the options provided with the tool and was wondering how I could set up something like this:
Screen my human data against potential contaminants (EColi, Yeast, Adapters,..) and only remove the hits that are classified as 'one-hit/one-library' AND 'multiple-hits/one-library'. I see that this feature is built-in as part of the plots, but was not clear if it could be (and how to) set up.

Thanks
SK

[cjdoherty]

There isn't a publication for fastq_screen. We recommend just citing the project URL.

Thank you. Will do!

[simonandrews]

Just want to make sure I'm not missing a publication, is there a preferred way to cite FastQ screen?
The program was so helpful we really appreciate it.
Thanks!

There isn't a publication for fastq_screen. We recommend just citing the project URL.

[cjdoherty]

Citing FastX Screen

Just want to make sure I'm not missing a publication, is there a preferred way to cite FastQ screen?
The program was so helpful we really appreciate it.
Thanks!

[simonandrews]

I sent you a direct mail about this, but just so the information stays in the post, I think the problem here is that you are only specifying the path to the directory which contains your indices, and not the full path to the actual database. In this case it's a little confusing in that the name of the database and the name of the folder it's in are the same (which makes sense, but since it doesn't have to be like that you need to explicitly tell the program).

I think the fix is simply to append the database name to the end of the paths, so instead of:

/data/users/khademul/Bowtie-Prebuilt-Index/hg19

..you'd have

/data/users/khademul/Bowtie-Prebuilt-Index/hg19/hg19

[abmmki]

configure fastq_screen.config

Hi,

I would like to use fastq_screen against Drosophila, Human, Mouse, Ecoli genome. I have downloaded Bowtie Pre-Built Index files and corresponding genome sequence (single fasta file).

I have prepared config file as below, and run command like following .... but got error:

#-------- Config file:

BOWTIE /data/users/bin/bowtie
BOWTIE2 /data/users/bin/bowtie2-2.2.4

THREADS 12
DATABASE Drosophila /data/users/Bowtie-Prebuilt-Index/dme_ucsc BOWTIE
DATABASE Human /data/users/Bowtie-Prebuilt-Index/hg19 BOWTIE
DATABASE Mouse /data/users/Bowtie-Prebuilt-Index/mm9 BOWTIE
DATABASE Ecoli /data/users/Bowtie-Prebuilt-Index/e_coli BOWTIE

#--------------- Command

fastq_screen --threads 12 --aligner bowtie --bowtie "-m 2 -g 1 --butterfly-search" $fq/MT1.fq $fq/MT2.fq $fq/MT3.fq $fq/MT4.fq $fq/MT5.fq $fq/MT6.fq $fq/MT7.fq $fq/MT8.fq

#-------------- Error

Using fastq_screen v0.4.4

Reading configuration from '/data/users/bin/fastq_screen_v0.4.4/fastq_screen.conf'

Using '/data/users/bin/bowtie/bowtie' as bowtie path

Using 12 threads for searches

Skipping DATABASE 'Drosophila' since no bowtie index was found at '/data/users/Bowtie-Prebuilt-Index/dme_ucsc'

Skipping DATABASE 'Human' since no bowtie index was found at '/data/users/Bowtie-Prebuilt-Index/hg19'

Skipping DATABASE 'Mouse' since no bowtie index was found at '/data/users/Bowtie-Prebuilt-Index/mm9'

Skipping DATABASE 'Ecoli' since no bowtie index was found at '/data/users/Bowtie-Prebuilt-Index/e_coli'

No search libraries were configured at /data/users/bin/fastq_screen_v0.4.4/fastq_screen line 124.



## But I see that Bowtie Prebuilt Index files are present in above mentioned pathways ....... fol example:

ls /data/users/Bowtie-Prebuilt-Index/hg19

hg19.1.ebwt
hg19.2.ebwt
hg19.3.ebwt
hg19.4.ebwt
hg19.fa
hg19.rev.1.ebwt
hg19.rev.2.ebwt

# Final directory names as the prefix of the pre-built index names.So, this is not the issue disccued already.

# It shows that Bowtie Index and corresponding genome seq files are present in the directory. Also I used these Index files for mapping already without problem.

# I have GD::Graph installed properly.

thanks

[Brian Bushnell]

Zapages,

Have you considered BBSplit? It is based on BBMap, but designed for a slightly different role; specifically, decontaminating or binning reads from multiple organisms. It maps simultaneously to all references and outputs reads to one file per reference. Each output file will only get reads that map best to that reference. Depending on your ambiguity settings, reads from conserved regions will either be written to the files of ALL references they map to equally well, or just one, or discarded. The output is fasta or fastq.