[nkaushik]

There are commercial softwares e.g. CLCBio, DNAStar, NextGene etc. to analyse NGS data that can take fastQ format as input data just follow the manual.

[vishnuamaram]

Hi Bukowski, mastal / simon andrews

I hereby write you as you are experts and pioneers in the NGS Analysis.

1) i have whole genome seq. data of human lymphocytes done on illumina hiseq 2000 paired end 2X100bp read length

2) i had done FASTQC of my data and trimmed low quality bases using FASTX toolkit.

Q) i am struck to go further with analysis as i am not sure of how to perform indexing.
--> what does the indexing do exactly. do we need to consider the Chr un... files for indexing.
kindly let me know the downstream analysis steps with clarity.
Will be very thankful to you.

Vishnu.

[hanshart]

...
Q) i am struck to go further with analysis as i am not sure of how to perform indexing.
--> what does the indexing do exactly. do we need to consider the Chr un... files for indexing.
kindly let me know the downstream analysis steps with clarity.
Will be very thankful to you.
Vishnu.

Hi Vishnu,
The indexing itself is not that important for you, it is just an algorithm to store your reference in a nice data structure. Every aligner uses it's own indexing strategy based on the alignment strategy.
What you want to do next is the alignment/mapping of your trimmed reads to a reference. For this you need a program like STAR, TopHat, Bowtie 1/2, BWA, .... So, you have to decide which mapping program to use and which reference. It is up to you and your biological questions, whether you want to include unplaced contigs (chr_Un..., UCSC) in your reference. Finally you end up with a folder of FASTA files or a single FASTA file fully describing your reference chromosomes/contigs. This FASTA has to be converted to an index suitable for your mapping program. Most often it should be only a single command to to this conversion step. For instance in Bowtie you simply type:

bowie-build REF_FASTA IDX_FOLDER/IDX_NAME

where REF_FASTA is the path to your reference FASTA (files).
For every mapping with Bowtie against this reference you have to provide the path to this index, as it is in fact just another representation of your reference FASTA.
Hope this helps

[vishnuamaram]

Hi hanshart,

Thank you very much for your instant response and I apologize for my delayed response.

At present, i am done with the indexing and alignment of my data using BWA. ( whole genome sequence of human lymphocytes).

I am interesting in looking for SNP's and if possible structural variations- indels, CNV's.

Other than SAMTOOLS, what other software tools may be required for the further downstream analysis. Kindly let me know.

Thank you,
Vishnu.

[mastal]

How to read FASTQ files?

Other popular tools are GATK from the Broad institute for finding SNPs/genotype calling, and dindel or pindel for finding indels.

[maivantan]

Dear all,
I am a new user of bwa.
however, i don't know the command on terminal how to read the raw data.
any one can help?
Thank you

[mastal]

Have a look at the Unix tutorial at the link below:

http://www.ee.surrey.ac.uk/Teaching/Unix/

commands like head, tail, more, less,

are useful for looking at large files.

[maivantan]

can any one type the full command that open one fastq file?
I can't open a fast file

[dpryan]

can any one type the full command that open one fastq file?
I can't open a fast file

Define what you mean by "open". To see the first few lines "head foo.fastq" or "zcat foo.fastq.gz | head", depending on whether it's compressed, will work.

[maivantan]

I run Miseq, and have fast file format.
I already download bwa, but i don't know how to run bwa for my file.
I don't know command on terminal to run bwa for align sequence.
can you help me?

[dpryan]

There are examples in the synopsis section of the bwa manual.

[maivantan]

i known these command but i could not run it.

[dpryan]

"could not run it" isn't enough information for anyone to help you with anything ever. We don't read minds here, tell us what happens when you try to run it and what you typed.

[maivantan]

i download and install bwa, the try to run using the command "bwa index ref.fa" in terminal, i found the no such directory or file as following:
USER-no-MacBook-Pro-4:bwa-0.7.5a user$ bwa index ref.fa
-bash: bwa: command not found

i have to put same directory of bwa and ref.fa file. but i don't how to do it.

can you have some suggestions please?

[Bukowski]

If you're in the directory with bwa in it, try this:

chmod 755 bwa
./bwa

I bet that solves at least part of the problem