[sabbir_barj]

Hi Nareshvasani,
Thanks a lot. You said before that several steps like trim, duplication removal from each fastq files and finally combining all fasta files into one fasta file. If you follow the second step, then how can you got several files and how can you merge file? Because you run the tools one after and working with output file of each tool?

Regards
Sabbir

[nareshvasani]

Hi Sabbir,


This cmd I used for my data. You do not have to follow same steps but you have to use this cmd as your reference as per your data need [as said bu Genomax]

I think you have not understand how this fastx toolkit work. You need to read manual of this toolkit properly.

First method you described it doesn't make any sense.
I used second method.

Best,
Nareshvasani

Hi NareshVasani,

Thanks for your kind reply. Did you run different trimming tools on the same file and the merge the output files of each tools or you run one after another?
Such as,
sample file >fastq_quality_filter >output file1
sample file> fastq_quality_trimmer> ouput file 2
sample file > fastx_trimmer> output file3
sample file > fastx_collapser >output file4

then merge the all 4 files.

then merge the four files, or
You run one tools one after, Like first you run fastq_quality_filter and then run fastq_quality_trimmer with output of fastq_quality_filter (input file is fastq_quality_filter output) and the following the tools.

If you followed first one then I want to how to you merge the files and why you did not use the fastq file?

Regards
Sabbir

[sabbir_barj]

Hi NareshVasani,

Thanks for your kind reply. Did you run different trimming tools on the same file and the merge the output files of each tools or you run one after another?
Such as,
sample file >fastq_quality_filter >output file1
sample file> fastq_quality_trimmer> ouput file 2
sample file > fastx_trimmer> output file3
sample file > fastx_collapser >output file4

then merge the all 4 files.

then merge the four files, or
You run one tools one after, Like first you run fastq_quality_filter and then run fastq_quality_trimmer with output of fastq_quality_filter (input file is fastq_quality_filter output) and the following the tools.

If you followed first one then I want to how to you merge the files and why you did not use the fastq file?

Regards
Sabbir

[GenoMax]

@sabbir: You are probably doing this already but use the example command lines/setting supplied by Naresh as a guideline. You will have to experiment with your own data.

What is your expected genome size? Do you have an idea of the approximate fold coverage you have? If there is a reference genome available then alignment may be a better option to try than assembly.

[nareshvasani]

Hi Sabbir,

Below are the following steps I used for trimming:

The following command keep reads which has quality score above 20 in at least 50% of bases.
> fastq_quality_filter -Q33 -q20 -p 50 -i <SAMPLE_NAME>.fastq -o <SAMPLE_NAME>.qulaity_filter.fastq

The following operation removes nucleotides having quality scores lower than 20 from the ends of reads. Furthermore, any trimmed reads having lengths less than 50 nucleotides are discarded altogether:
> fastq_quality_trimmer -Q33 -t 20 -l 50 -i <SAMPLE_NAME>.fastq -o <SAMPLE_NAME>.clean.fastq

To remove base sequence content and GC content from the end of reads, following command was used. It removes 15 nucleotides from the end of reads.
> fastx_trimmer –Q33 –f 1 -l 335 -i <SAMPLE_NAME>.clean.fastq -o <SAMPLE_NAME>.fastx_trimmer.fastq

After this step, the read length distribution changed minimally, with the majority of reads retaining their full length. In addition around 25% of the reads were discarded completely.

In order to remove identical sequences, fastx_collapser tool was used:
> fastx_collapser -v -i <SAMPLE_NAME>.fastq -o <SAMPLE_NAME>_collapsed.fasta
Above tools removes few millions reads from each files while maintaining all read counts and gives output in fasta format.


##All of the above steps will help you to reduce size of input file and improve quality of each fastq file.



Best,
NareshVasani

[sabbir_barj]

Hi nareshvasani,

Thanks for your reply. I got huge number of contigs from the three assembler than I expected. From the genome sequences of my species, the contigs number should be 30000-40000 but I got more than 200000 contigs. Also I got large size of trancripts than I expected. Following pipeline I used for assembl
1. remove adapter by cutadapt
2. remove duplicate
3. then assembly with trinity.
I also used trinity quality options

I have also a big input file (Fastq). Accroding to your suggestions I need to run several tools to improve input file. Can you please tell me in details the several steps like trim, duplication removal from each fastq files and finally combining all fasta files into one fasta file for assembly (name of different tools and how to combine the fasta files)?

Regards
Sabbir

[nareshvasani]

Hi Sabbir,

How did you confirm that assembly from MIRA4, Trinity and velvet assembler were not good enough?
I had very big input file (fastq) which was not very well handled by all assembler. So, in order to generate input file which can be very well handled by Trinity, i processed several steps like trim, duplication removal from each fastq files and finally combining all fasta files into one fasta file.


Below is the command I used for Trinity [I used same cmd as suggested by trinity website]

1] Trinity.pl -seqType fa -min_contig_length 200 -JM 40G -CPU 4 -single inputfilename.fasta -output trinity_output

#### took 2 days to complete 100% ######

2] /bin/util/TrinityStats.pl Trinity.fasta # gives basic stat information i.e.e assembly file info.

Total trinity transcripts: 128578
Total trinity components: 43455
Contig N50: 862


Hope this helps.
Best luck,
Naresh

Hi nareshvasani,

I am curious about your assembly result with trinity. I have a instrument of Ion proton data and I am trying to assembly the transcriptome data. But I did not get good result with MIRA4, Trinity and Velvet assembler. Can you tell me your command line for trinity and general information about your raw read and assembly file? it will help me a lot.Thanks


sabbir

[sabbir_barj]

Hi nareshvasani,

I am curious about your assembly result with trinity. I have a instrument of Ion proton data and I am trying to assembly the transcriptome data. But I did not get good result with MIRA4, Trinity and Velvet assembler. Can you tell me your command line for trinity and general information about your raw read and assembly file? it will help me a lot.Thanks


sabbir

Hi,

Thanks for your prompt reply.
I really appreciate your suggestion.
Do you think if I put some more input option for butterfly, inchworm, kmer and Chrysalis, it will give me better contig file?

Thanks,
Naresh

[nareshvasani]

Hi

Hi everyone,

I run below cmd for alignment.
bin/util/alignReads.pl --seqType fq --single CombineIonXpressRNA_010_NareshPool_Chip1_2_WT2_fastxtrimmer_from_quality_trimmer.fastq --target /media/DATAPART3/Combine_Files/Velvetoptimiser_27_37/trinity_output/Trinity.fasta --aligner bowtie --retain_intermediate_files --output align_bowtie_output1

##output in terminla show as follow:

CMD: bowtie -k 20 -S --sam-nohead -q target single_fa > single_fa.pre.sam
# reads processed: 18001999
# reads with at least one reported alignment: 3881611 (21.56%)
# reads that failed to align: 14120388 (78.44%)
Reported 6874538 alignments to 1 output stream(s)
CMD: touch single_fa.pre.sam.finished
CMD: /root/Trinity/util/../util/SAM_filter_out_unmapped_reads.pl single_fa.pre.sam > single_fa.sam
-filtered 14120388 of 20994926 reads as unaligned = 67.26% unaligned reads
CMD: touch single_fa.sam.finished
CMD: sort -T . -S 2G -k 1,1 -k 3,3 single_fa.sam > single_fa.nameSorted.sam
CMD: touch single_fa.nameSorted.sam.finished
-child alignment process completed.

## Alignment steps succeeded.

CMD: sort -T . -S 2G -k 3,3 -k 4,4n single_dir/single_fa.nameSorted.sam > single_dir/single.coordSorted.sam
CMD: touch single_dir/single.coordSorted.sam.finished
CMD: cp single_dir/single.coordSorted.sam align_bowtie_output1.pre.coordSorted.sam
CMD: touch align_bowtie_output1.pre.coordSorted.sam.finished
CMD: cp align_bowtie_output1.pre.coordSorted.sam align_bowtie_output1.coordSorted.spliceAdjust.sam
CMD: touch align_bowtie_output1.coordSorted.spliceAdjust.sam.finished
CMD: cp /media/DATAPART3/Combine_Files/Velvetoptimiser_27_37/trinity_output/align_bowtie_output1/align_bowtie_output1.coordSorted.spliceAdjust.sam align_bowtie_output1.coordSorted.sam
CMD: touch align_bowtie_output1.coordSorted.sam.finished
CMD: samtools view -bt target.fa.fai -S align_bowtie_output1.coordSorted.sam | samtools sort - align_bowtie_output1.coordSorted
[bam_header_read] EOF marker is absent. The input is probably truncated.

[sam_header_read2] 102085 sequences loaded.
[bam_sort_core] merging from 3 files...
CMD: touch align_bowtie_output1.coordSorted.bam.finished
CMD: sort -T . -S 2G -k 1,1 -k 3,3 align_bowtie_output1.coordSorted.sam > align_bowtie_output1.nameSorted.sam
CMD: touch align_bowtie_output1.nameSorted.sam.finished
CMD: samtools view -bt target.fa.fai align_bowtie_output1.nameSorted.sam > align_bowtie_output1.nameSorted.bam
[sam_header_read2] 102085 sequences loaded.
CMD: samtools index align_bowtie_output1.coordSorted.bam

As per above ouput:
20994926 reads where used for alignment. But in actually I have only 18,001,199 reads.
# 67.26% was unaligned and 32.74% was aligned

So Questions are:
1] Why it used more amout of input reads i.e. is 20 million instead of 18M.
2] 32% is cosidered as good or bad alignment? If bad, how do i improve alignment %
3] What does this mean == EOF marker is absent. The input is probably truncated


Thanks in advance.
Naresh

[nareshvasani]

Hi Genomax,

Thanks for feedback.

I don't have paired end data.
I was confused about output of alignReads.pl script. i.e. how to confirm percentage of alignment. That's why asked about pairing. Sorry about that.
Can you please give me some feedback about how to check preservation of assembly and accurary of assembled contigs.

Thanks,
Naresh

Bowtie 2 supports gapped, local, and paired-end alignment modes. That may be a reason to prefer it over bowtie.

Do you have paired-end data? Otherwise how will you get information about pairing?

[GenoMax]

Bowtie 2 supports gapped, local, and paired-end alignment modes. That may be a reason to prefer it over bowtie.

Do you have paired-end data? Otherwise how will you get information about pairing?

[nareshvasani]

Thanks,

If I use bowtie, does it make any difference in alignment.
When I am running cmd with bowtie, while processing it shows below message:
CMD: bowtie -k 20 -S --sam-nohead -q target single_fa > single_fa.pre.sam

Upto my understanding it says sam has no header. Am I correct?

when i run stat.pl cmd to obtain read alignment statistics:
result shows:
read_type count pct
single 6874526 100.00

but no information about proper and improper pairing.

Hope you can shed lights on this.

Thanks a bunch.
Naresh

I am surprised that Trinity would be looking for tophat2. However bowtie2 is closely associated with tophat2. I suggest installing tophat2. You may never need it but that should be the way to get Trinity to use bowtie2.

[westerman]

I am surprised that Trinity would be looking for tophat2. However bowtie2 is closely associated with tophat2. I suggest installing tophat2. You may never need it but that should be the way to get Trinity to use bowtie2.

[nareshvasani]

Hi westerman,

Hi westerman,

Thanks for you reply.
fullpath was mising in my cmd line.

Above cmd worked but with bowtie not with bowtie2.

With below cmd:
/bin/util/alignReads.pl --seqType fq --single CombineIonXpressRNA_010_NareshPool_Chip1_2_WT2_fastxtrimmer_from_quality_trimmer.fastq --target /media/DATAPART3/Combine_Files/Velvetoptimiser_27_37/trinity_output/Trinity.fasta --aligner bowtie2 --retain_intermediate_files --num_top_hits 20 --output align_bowtie_output1

Following error:
which: no tophat2 in (/root/perl5/bin:/usr/local/sbin:/usr/local/bin:/sbin:/bin:/usr/sbin:/usr/bin:/root/bin:/root/Trinity/util/../trinity-plugins/rsem/sam/)
Error, path to required tophat2 cannot be found at /bin/util/alignReads.pl line 234.

#If I used bowtie instead of bowtie 2, it work fine with problem i.e. sam file has no header.

Hope you can help me out.
Thanks in advance.
Naresh

Well, off-hand I would say that you need to give the whole path to the Trinity.fasta file. I suspect that it is not located in the directory in which you are located.

In these 'file not found' cases it is always helpful to the rest of us if you can include the results of:

pwd

and a

ls -l

[westerman]

Well, off-hand I would say that you need to give the whole path to the Trinity.fasta file. I suspect that it is not located in the directory in which you are located.

In these 'file not found' cases it is always helpful to the rest of us if you can include the results of:

pwd

and a

ls -l