Tag Archives: assembly

Genome Assembly – Olympia oyster Redundans/Canu vs. Redundans/Racon

Decided to compare the Redundans using Canu as reference and Redundans using Racon as reference. Both reference assemblies were just our PacBio data.

Jupyter notebook (GitHub): 20171005_docker_oly_redundans.ipynb

Notebook is also embedded at the end of this post.

Results:

It should be noted that the paired reads for each of the BGI mate-pair Illumina data did not assemble, just like last time I used them:

  • 160103_I137_FCH3V5YBBXX_L3_WHOSTibkDCABDLAAPEI-62_2.fq.gz
  • 160103_I137_FCH3V5YBBXX_L3_WHOSTibkDCACDTAAPEI-75_2.fq.gz
  • 160103_I137_FCH3V5YBBXX_L4_WHOSTibkDCABDLAAPEI-62_2.fq.gz
  • 160103_I137_FCH3V5YBBXX_L4_WHOSTibkDCACDTAAPEI-75_2.fq.gz
  • 160103_I137_FCH3V5YBBXX_L5_WHOSTibkDCAADWAAPEI-74_2.fq.gz
  • 160103_I137_FCH3V5YBBXX_L6_WHOSTibkDCAADWAAPEI-74_2.fq.gz

Redundans with Canu is better, suggesting that the Canu assembly is the better of the two PacBio assemblies (which we had already suspected).

QUAST comparison using default settings:

Interactive link:http://owl.fish.washington.edu/Athaliana/quast_results/results_2017_10_06_22_21_06/report.html

QUAST comparison using –scaffolds setting:

Interactive link: http://owl.fish.washington.edu/Athaliana/quast_results/results_2017_10_06_22_27_26/report.html

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Genome Assembly – minimap/miniasm/racon Overview

Previously, I used the following three tools to do quick assembly of our Olympia oyster PacBio data:

I’m just posting this quick overview to make it easier to follow what was actually done without having to read through three different notebook entries and corresponding Jupyter notebooks.

When I say “quick assembly”, I mean it. The entire assembly process probably takes about an hour on the computer I used – that seems fast.

Here’s the quick and dirty of what was done:

1 Run minimap:

This uses a pre-built set of defaults (the ava-pb in the code below) for analyzing PacBio data. Minimap only accepts two FASTQ files and you need to map your FASTQ file against itself. So, if you have multiple FASTQ sequencing files, you have to concatenate them into a single file prior to running minimap.

minimap2 -x ava-pb -t 23 
20170911_oly_pacbio_cat.fastq 
20170911_oly_pacbio_cat.fastq 
> 20170911_minimap2_pacbio_oly.paf

2 Run miniasm:

This uses your concatenated FASTQ file and the PAF file output from the miniasm step. The code below is taken from the example provided in the miniasm documentation; there are other options available.

miniasm 
-f 
/home/data/20170911_oly_pacbio_cat.fastq /home/data/20170911_minimap2_pacbio_oly.paf > /home/data/20170918_oly_pacbio_miniasm_reads.gfa

3 Convert miniasm output GFA to FASTA

The FASTA file is needed to re-run minimap in Step 4 below.

awk '$1 ~/S/ {print ">"$2"n"$3}' 20170918_oly_pacbio_miniasm_reads.gfa > 20170918_oly_pacbio_miniasm_reads.fasta

4 Run minimap with default settings

Using the default settings maps the FASTQ reads back to the contigs (the PAF file) created in the fist step. These mappings are required for Racon assembly (Step 5).

minimap2 
-t 23 
20170918_oly_pacbio_miniasm_reads.fasta 20170905_minimap2_pacibio_oly.paf > 20170918_minimap2_mapping_fasta_oly_pacbio.paf

5 Run racon

The output file is the FASTA file listed below.

racon -t 24 
20170911_oly_pacbio_cat.fastq 
20170918_oly_pacbio_minimap_mappings.paf 
20170918_oly_pacbio_miniasm_assembly.gfa 
20170918_oly_pacbio_racon1_consensus.fasta
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Data Received – Initial Geoduck Genome Assembly from BGI

The initial assembly of the Ostrea lurida genome is available from BGI. Currently, we’ve stashed it here:

http://owl.fish.washington.edu/P_generosa_genome_assemblies_BGI/20160314/

The data provided consisted of the following three files:

  • md5.txt
  • N50.txt
  • scaffold.fa.fill

md5.txt – Checksum file to verify integrity of files after downloading.

N50.txt – Contains some very limited stats on scaffolds provided.

scaffold.fa.fill – A FASTA file of scaffolds. Since these are scaffolds (and NOT contigs!), there are many regions containing NNNNNN’s that have been put in place for scaffold assembly based on paired-end spatial information. As such, the N50 information is not as useful as it would be if these were contigs.

Additional assemblies will be provided at some point. I’ve emailed BGI about what we should expect from this initial assembly and what subsequent assemblies should look like.

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Data Received – Initial Olympia oyster Genome Assembly from BGI

The initial assembly of the Ostrea lurida genome is available from BGI. Currently, we’ve stashed it here:

http://owl.fish.washington.edu/O_lurida_genome_assemblies_BGI/20160314/

The data provided consisted of the following three files:

  • md5.txt
  • N50.txt
  • scaffold.fa.fill

md5.txt – Checksum file to verify integrity of files after downloading.

N50.txt – Contains some very limited stats on scaffolds provided.

scaffold.fa.fill – A FASTA file of scaffolds. Since these are scaffolds (and NOT contigs!), there are many regions containing NNNNNN’s that have been put in place for scaffold assembly based on paired-end spatial information. As such, the N50 information is not as useful as it would be if these were contigs.

Additional assemblies will be provided at some point. I’ve emailed BGI about what we should expect from this initial assembly and what subsequent assemblies should look like.

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Second look at Geoduck transcriptome

Last week I popped out a quick assembly and annotation on our geoduck gonadal transcriptome. A second assembly was also done using Trinity.


Updates
August 3 – Confirmed // in file location had no impact on assembly.
July 14 – TransDecoder protein annotations
10:40am – added TransDecoder results
10:29am – added Stats via Trinity


Trinity.pl 
--seqType fq 
-JM 24G 
--left /Volumes/web/cnidarian/Geo_Pool_F_GGCTAC_L006_R1_001_val_1.fq /Volumes/web/cnidarian/Geo_Pool_M_CTTGTA_L006_R1_001_val_1.fq 
--right /Volumes/web/cnidarian//Geo_Pool_F_GGCTAC_L006_R2_001_val_2.fq /Volumes/web/cnidarian//Geo_Pool_M_CTTGTA_L006_R2_001_val_2.fq 
--CPU 16 

trinity_out_dir_1B54203C.png

Output

0:999   127840
1000:1999   18164
2000:2999   5321
3000:3999   1817
4000:4999   762
5000:5999   291
6000:6999   135
7000:7999   73
8000:8999   22
9000:9999   29
10000:10999     4
11000:11999     5
12000:12999     3
13000:13999     4
14000:14999     4
15000:15999     3
16000:16999     0
17000:17999     2
18000:18999     1

Total length of sequence:   101862868 bp
Total number of sequences:  154480
N25 stats:          25% of total sequence length is contained in the 8095 sequences >= 2045 bp
N50 stats:          50% of total sequence length is contained in the 26158 sequences >= 1014 bp
N75 stats:          75% of total sequence length is contained in the 64574 sequences >= 446 bp
Total GC count:         37657770 bp
GC %:               36.97 %
hummingbird:Geo-trinity steven$ /Users/gilesg/compile/trinityrnaseq_r20131110/util/TrinityStats.pl /Volumes/web/cnidarian/Geo-trinity/trinity_out_dir/Trinity.fasta 


################################
## Counts of transcripts, etc.
################################
Total trinity transcripts:  154480
Total trinity components:   100155
Percent GC: 36.97

########################################
Stats based on ALL transcript contigs:
########################################

    Contig N10: 3444
    Contig N20: 2385
    Contig N30: 1766
    Contig N40: 1343
    Contig N50: 1014

    Median contig length: 371
    Average contig: 659.39
    Total assembled bases: 101862868


#####################################################
## Stats based on ONLY LONGEST ISOFORM per COMPONENT:
#####################################################

    Contig N10: 2999
    Contig N20: 2026
    Contig N30: 1462
    Contig N40: 1067
    Contig N50: 768

    Median contig length: 321
    Average contig: 553.88
    Total assembled bases: 55473621

Rerunning to see if double slash was a problem- did not see anything in error. Also running TransDecoder


TransDecoder Results

Ran the following

/Users/gilesg/compile/trinityrnaseq_r20131110/trinity-plugins/TransDecoder_r20131110/TransDecoder -t  /Volumes/web/cnidarian/Geo-trinity/trinity_out_dir/Trinity.fasta

This provided a peptide file with 36003 sequences.

!head /Volumes/web-1/cnidarian/Geo-trinity/Trinity.fasta.transdecoder.pep

>cds.comp100047_c0_seq2|m.5982 comp100047_c0_seq2|g.5982 ORF comp100047_c0_seq2|g.5982 comp100047_c0_seq2|m.5982 type:internal len:142 (-) comp100047_c0_seq2:3-425(-)
NAECRDLYKIFTQILSVRSQEGKIVIPDEFATKIRNWLGNKEELFKEAHNQKIITFYNEY
TREENTFNPIRGKRPMSVPDMPERKYIDQLSRKTQSQCDFCKYKTFTAEDTFGRIDSNFS
CSASNAFKLDHWHALFLLKTH


Running blastp on Trinity.fasta.transdecoder.pep

!blastp 
-query /Volumes/web/cnidarian/Geo-trinity/Trinity.fasta.transdecoder.pep 
-db /usr/local/bioinformatics/dbs/uniprot_sprot.fasta 
-evalue 1e-5 
-max_target_seqs 1 
-max_hsps 1 
-outfmt 6 
-num_threads 4 
-out /Volumes/web/cnidarian/Geo-trinity/Trinity.fasta.transdecoder.pep-blastp-uniprot-2.out

results: http://eagle.fish.washington.edu/cnidarian/Geo-trinity/Trinity.fasta.transdecoder.pep-blastp-uniprot-2.out

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Transcriptome Assembly

 

Trinity

################################
## Counts of transcripts, etc.
################################
Total trinity 'genes':  117729
Total trinity transcripts:  145222
Percent GC: 40.87

########################################
Stats based on ALL transcript contigs:
########################################

    Contig N10: 3392
    Contig N20: 2262
    Contig N30: 1685
    Contig N40: 1268
    Contig N50: 946

    Median contig length: 335
    Average contig: 617.51
    Total assembled bases: 89676218


#####################################################
## Stats based on ONLY LONGEST ISOFORM per 'GENE':
#####################################################

    Contig N10: 3252
    Contig N20: 2047
    Contig N30: 1465
    Contig N40: 1065
    Contig N50: 767

    Median contig length: 305
    Average contig: 547.96
    Total assembled bases: 64511364


#!/bin/bash

TRIN="/home/ggoetz/compile/trinityrnaseq_r20140413p1"

export PATH=~/compile/rsem-1.2.3/sam:${PATH}

${TRIN}/Trinity 
    --seqType fq 
    --JM 44G 
    --left left.fq 
    --right right.fq 
    --CPU 6 
    --normalize_reads 
    --min_kmer_cov 2 
    --quality_trimming_params 
    "LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLENGTH:36"
In [3]:
!head //Volumes/web/cnidarian/SeaStar/trinity_assemblies/run1/Trinity.fasta
>c2_g1_i1 len=233 path=[20:0-32 20:33-65 20:66-98 52:99-232]

TCTTGGTCTTGGACGTGGACTTGCTGGTCTTGGTCTTGGTCTTGGACGTGGACTTGCTGG

TCTTGGTCTTGGTCTTGGACGTGGACTTGCTGGTCTTGGTCTTGTTCTTGGTCTTGTTCT

TTGTCTTGTTCTTGTTCTTGTTTATGTCCTTGTTTAGGGTTGTTGTTGGGTTTGTTGCTG

TGTTTTGGCGGGTTGTTGTTGTTTTGGGGGTTTTGGTTGTTTGTTTGTTTGTG

>c108_g1_i1 len=239 path=[1:0-131 133:132-238]

CACTTCGTATATGCTTTATAGACTTCTTGTACGATGTAAAACTCAGACTTTTAAAATCTT

TTCTCATTTTTTGTAAAACTTTATAGAATAATTTTTTCTCTCTTGGGATATATCTACACT

TTCAACTTGCTTAAAAAAAATATAGATAGTGTATGGTGTATGGAGGATTGTGTATTTCAC

ATGTGAGGTACTGTGTTACTAAATTTAGTTGTCGTGACAGAGAGAGGAACAGAGCAGGG


In [5]:
!fgrep -c ">" /Volumes/web/cnidarian/SeaStar/trinity_assemblies/run1/Trinity.fasta
145222


In [6]:
!fgrep -c ">" /Volumes/web/cnidarian/SeaStar/trinity_assemblies/run2/Trinity.Cufffly.fasta
160038


CLC

Trimming

greenbird_1977290F.png

de novo assembly

greenbird_19772A25.png

summary stats

greenbird_19772955.png

In [8]:
!head /Volumes/web/cnidarian/SeaStar_transc_v2.fa
>3291_5903_10007_H94MGADXX_V_CF71_ATCACG_R1_(paired)_trimmed_(paired)_contig_1

CAAATATATGAACGGTTGATTGTCAACGATTAGTACATGTTTTCATTGTTCCCCACGCCC

GCCCCCCCCCACTCAAACATTTAAAGTGTGAAATATTATTTATCCACAAATTTCCTTAAA

CCTGCAAACTTGTCTGCTGTCTCTTATTGGAAGTTATGAAAAAGAACAACGGGTTTTCTT

TAAAGGGTCTGCGTGCGATTTTCAACCTTTTGAGTAATAGCAGTTATTTTGATAACCGAT

TTTTTTCAAAGCTCAACAGCTTTTTAAAATAAGGAATCCTATAATGGCCAAACGAATACT

ATAAAAATAAGGGTTCTCTTAATTGTATAAAACGTATAATTTTATCAATTTTGGGACCGT

GTAATTTTTTAAAGACCACAAGAATGTTACATACAACAAATAGACGAAACTCGTAGCTTT

GGAAACTACGTCATGGGCGTTTGGTCAAAAGCTGGAGAGAAAGAGAGGTGGGGTGCCAGA

CTTAAGTAGTCACGTGATCTGACCAACGCACATCGGAAGCTCGATCGGATGAAATCTTCT


In [9]:
!fgrep -c ">" /Volumes/web/cnidarian/SeaStar_transc_v2.fa
30578


In []:

 

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