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This article is about how pacbio uses Iso-Seq 3 for data analysis. The editor thinks it is very practical, so share it with you as a reference and follow the editor to have a look.

Pacbio third-generation full-length transcriptome data analysis process Iso-Seq 3

Building database by Iso-Seq

There are three types of database building schemes for Iso-Seq:

The whole library is a full-length transcriptome of samples, there is no need to add barcode to distinguish samples.

The full-length transcriptome of different samples, plus different barcode, can be put together for database sequencing.

Some targeted genes can also be sequenced for full-length transcriptome.

Iso-Seq 3 for data analysis

Analysis of full-length transcriptome by Iso-Seq3

1 ccs (Circular Consensus Calling)

Ccs acquires consistent sequences, requiring that each sequenced reads contains at least one end of the primer sequence.

Ccs test.subreads.bam ccs.bam-noPolish-minPasses 1

2 removal of sequencing primers and barcode

This step is done by using lima, which is also the latest official program, and its speed and accuracy have been greatly improved compared with previous algorithms. It can also distinguish different samples based on barcode sequence.

Lima ccs.bam barcoded_primers.fasta demux.ccs.bam-isoseq-no-pbi

3 clustering (cluster)

Clustering is completed by IsoSeq3 software, this step will first remove the ployA tail, and remove the link structure, and then cluster similar sequences, preferably to form a full-length reads.

Isoseq3 cluster demux.primer_5p--primer_3p.bam unpolished.bam-verbose

4 polishing (polish)

This step is mainly to merge the transcripts of clustering into a complete consistent sequence.

Isoseq3 polish-j 16 unpolished.bam test.subreads.bam polished.bam Thank you for your reading! This is the end of this article on "how pacbio uses Iso-Seq 3 for data analysis". I hope the above content can be of some help to you, so that you can learn more knowledge. if you think the article is good, you can share it for more people to see!

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