HIFI-BARCODES-PACBIO-PIPELIN User's Guide V1.0 20170707

DESCRIPTION

HIFIBarcode is used to produce full-length COI barcodes from pooled PCR amplicons generated by individual specimens.

INSTALLATION

• Clone from github

$ git clone https://github.com/comery/HIFI-barcode-pacbio.git

• Download a ZIP file Go to website https://github.com/comery/HIFI-barcode-hiseq and click 'Download ZIP'

Requirements

(1) software

• PicBio smrtanalysis | http://www.pacb.com/products-and-services/analytical-software/smrt-analysis/

(2) programming language

• standard perl
• standard python(python2 is ok)

(3) perl module

• Bio::Perl( exactlly Bio::Seq )

(4) main perl and python scripts in bin/

• 1.primer_like_extract.pl
• 2.cluster_count_passes_length.pl
• ccs_passes.py | look => here| or directly download => here
• fish_ccs.pl

DATA requirements:

(1) pacbio original H5 file input

• 01.data/*.h5 ( linkage will be available soon )

(2) primers list

• experiment_data/primer.lst

  for GGTCAACAAATCATAAAGATATTGG
  rev TAAACTTCAGGGTGACCAAAAAATCA

(3) index(barcodes for identifying samples) list

• experiment_data/index.xls

  001 AAAGC
  002 AACAG
  003 AACCT
  004 AACTC
  005 AAGCA
  ... .....

(4) samples_location.tab

• samples name and corresponding location in 96-cell plate

  1 A01
  2 B01
  3 C01
  4 D01
  5 E01
  . ...

Overview of steps

If you installed PacBio smrtanalysis, I suppose you get the file path of setup.sh, more about Pacbio Data : http://www.pacb.com/wp-content/uploads/SMRT-Link-User-Guide-v4.0.0.pdf

e.g: setup_path='/path/PicBio/smrtanalysis/current/etc/setup.sh'

step 1 extract CCS from h5 files

Input:

• my_inputs.fofn

Output:

• log

• data *.ccs.fasta *.ccs.fastq *.ccs.h5 reads_of_insert.fasta reads_of_insert.fastq slots.pickle

• workflow

• results

my_inputs.fofn contains files list of Pacbio H5 file in 01.data/, like this: ./01.data/m170506_092957_42199_c101149142550000001823255607191735_s1_p0.1.bax.h5 ./01.data/m170506_092957_42199_c101149142550000001823255607191735_s1_p0.bas.h5 ./01.data/m170506_092957_42199_c101149142550000001823255607191735_s1_p0.3.bax.h5 ./01.data/m170506_092957_42199_c101149142550000001823255607191735_s1_p0.2.bax.h5

run:

$ source /path/PicBio/smrtanalysis/current/etc/setup.sh
$ fofnToSmrtpipeInput.py my_inputs.fofn > my_inputs.xml
$ smrtpipe.py --params=settings.xml xml:input.xml

step 2 extract passes number from CCS h5 files

Input:

• /data/*.ccs.h5

Output:

• ccs_passes.lst

run: in sure that you have run source /path/PicBio/smrtanalysis/current/etc/setup.sh

$ python bin/ccs_passes.py  data/*.ccs.h5 >ccs_passes.lst

step 3 filtering CCS by passes number (>15)

Input:

• ccs_passes.lst
• data/reads_of_insert.fasta

Output:

• ccs_passes_15.fa

run:

$ awk '$2>=15{print $1}' ccs_passes.lst >ccs_passes_15.lst
$ perl ./bin/fish_ccs.pl ccs_passes_15.lst data/reads_of_insert.fasta >ccs_passes_15.fa

step 4 assigning CCS to samples by index

Input:

• experiment_data/primer.fa
• experiment_data/index.xls
• ccs_passes_15.fa

Output: "outdir" name is up to you, here default value is "02.assignment"

02.assignment/

• assign.log.txt
• ccs.successfully_assigned.fa
• check.ccs_passes_15.fa.log

run:

$ perl ./bin/1.primer_like_extract.pl -p experiment_data/primer.fa -index experiment_data/index.xls -fa ccs_passes_15.fa -cm 2 -cg 1 

step 5 clustering CCS of each sample to find best one

Input:

• ccs.successfully_assigned.fa
• check.ccs_passes_15.fa.log
• ccs_passes.lst

Output:

• cluster.top1.fas
• cluster.id.txt
• cluster.all.fa

run:

$ cd 02.assignment/
$ perl ../bin/2.cluster_count_passes_length.pl -ccs ccs.successfully_assigned.fa -pattern check.ccs_passes_15.fa.log -passes ../ccs_passes.lst
$ perl ../bin/change_name-location.pl cluster.top1.fas >hifi-barcode-pacbio.cluster.top1.fa

ALL DONE!

So, "hifi-barcode-pacbio.cluster.top1.fa" is final result!

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ACKNOWLEDGEMENT

Thanks Hailin Pan for inspiring me about dynamic programming in script-"1.primer_like_extract.pl", I did learn much from that!

CONTACT US

Email: yangchentao at genomics dot cn

CITATION

Liu, Shanlin, Chentao Yang, Chengran Zhou, and Xin Zhou. "Filling reference gaps via assembling DNA barcodes using high-throughput sequencing–moving toward barcoding the world." GigaScience(2017).

LATEST RELEASE

Version 1.0 201707