What is the next step after download the ncbi sra data of pacbio RSII sequencing

Question

[Sparrow_kop]

Hi, guys! I am a newbie in pabio sequence analysis , recently I download some pacbio RSII data from ncbi sra database, after fastq-dump , I get a fastq file which I think is the raw subread data.

But now I want to do ccs(Circular Consensus Sequence calling) step , the pacbio ccs binary software need the bam or bax (bax can be transformed to bam )file as input , also in the sra website , there is no h5 format file submitted by submitter , so there only has fastq file , so I don't know how to do with the pacbio fastq file ? Thanks!

Answer 1

[harish]

Since you say that you are getting Fastq/fasta files, the first step is to correct them. The best way to go about it is to either use Falcon till the generation of preads or Canu's correct-trim mode or use proovread.

If you had the bax/bam files, this would have been easier as you could import them into SMRTlink and you could have used it's pushbutton nature :)

[Sparrow_kop]

Hi, harish, thanks! I will try it ! Also I had contacted the pacbio support , they said that the best solution is to get the raw bax/bam file......

Answer 2

[lakhujanivijay]

Hi Sparrow_kop

I don't know about "pacbio ccs binary software" but you can generate CCS using pabio SMRT link. Take a look at the consensus sequence resulting from alignment between subreads taken from a single ZMW.

Generation of the CCS read does not include or require alignment against a reference sequence. Generation of CCS reads using the CCS algorithm requires at least two full-pass subreads from the insert

Look at this video

[Sparrow_kop]

Hi Vijay Lakhujani , thank you for your answer! I watched the video in your link, but I found that the SMRT link program need the bam/RSII(which is .bax) format file as input ,So it don't support fastq file as input , do you used this program which import fastq file for analysis? thanks!