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AssemblyLog.v1.1.txt
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AssemblyLog.v1.1.txt
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## Content
# Aim and reference genome
# 1. Initial steps
# 2. possible deduplication and normalization steps before assembly
# 3. spades de novo assembly
# 4. blast against ref genome and use contigs as query
##############################
## Aim and reference genomes ##
"Obtain a full Pseudoalteromonas rubra genome"
Reference genome based on 16S rRNA gene blast result: Pseudoalteromonas rubra strain SCSIO 6842
All 5,913,780
Chromosome 1 4,532,889
Chromosome 2 1,311,023
Plasmid pMBL6842 69,868
https://www.ncbi.nlm.nih.gov/assembly/GCA_001482385.1
*NEW* Reference genome (12.01.2019): Pseudoalteromonas sp. R3
All 5,824,782
Chromosome 1 4,496,250
Chromosome 2 1,328,532
https://www.ncbi.nlm.nih.gov/assembly/GCA_004014715.1
######################
## 1. Initial steps ##
# Quality control
fastqc *.gz
multiqc .
# Sample Name % Dups % GC M Seqs
# NG-19286_1783_0s_lib326994_6367_2_1 22.0% 47% 4.5
# NG-19286_1783_0s_lib326994_6367_2_2 22.0% 47% 4.5
# NG-19286_1783_0s_lib326994_6377_2_1 31.4% 47% 3.8
# NG-19286_1783_0s_lib326994_6377_2_2 30.9% 47% 3.8
# merge libraries
cat *_1.fastq.gz > NG_1783_R1.fastq.gz
cat *_2.fastq.gz > NG_1783_R2.fastq.gz
#######################################################################
## 2. possible deduplication and normalization steps before assembly ##
# adapter and contamination removal
~/phylo/bbmap/bbduk.sh in1=NG_1783_R1.fastq.gz in2=NG_1783_R2.fastq.gz out1=NG_1783_R1_bbduk.fastq.gz out2=NG_1783_R2_bbduk.fastq.gz ref=~/phylo/bbmap/resources/adapters.fa ktrim=r k=23 mink=11 hdist=1 tpe tbo
BBDuk version 37.66
maskMiddle was disabled because useShortKmers=true
Initial:
Memory: max=1407m, free=1376m, used=31m
Added 216529 kmers; time: 0.372 seconds.
Memory: max=1407m, free=1310m, used=97m
Input is being processed as paired
Started output streams: 0.362 seconds.
Processing time: 500.187 seconds.
Input: 16560750 reads 2500673250 bases.
KTrimmed: 109614 reads (0.66%) 2898976 bases (0.12%)
Trimmed by overlap: 52638 reads (0.32%) 495056 bases (0.02%)
Total Removed: 50 reads (0.00%) 3394032 bases (0.14%)
Result: 16560700 reads (100.00%) 2497279218 bases (99.86%)
Time: 500.947 seconds.
Reads Processed: 16560k 33.06k reads/sec
Bases Processed: 2500m 4.99m bases/sec
# normalization
~/phylo/bbmap/bbnorm.sh in1=NG_1783_R1_bbduk.fastq.gz in2=NG_1783_R2_bbduk.fastq.gz out1=NG_1783_R1_bbduk_bbnorm.v2.fastq.gz out2=NG_1783_R2_bbduk_bbnorm.v2.fastq.gz target=100 min=5
################################
## 3. spades de novo assembly ##
spades.py -1 NG_1783_R1_bbduk_bbnorm.v2.fastq.gz -2 NG_1783_R2_bbduk_bbnorm.v2.fastq.gz -o spades_NG_1783_bbduk_norm_denovo_cf_v4 -t 8 -k 21,33,55,77,99,127 --careful
A C G T N IUPAC Other GC GC_stdev
0.2621 0.2365 0.2387 0.2627 0.0002 0.0000 0.0000 0.4752 0.0860
Main genome scaffold total: 116
Main genome contig total: 126
Main genome scaffold sequence total: 5.935 MB
Main genome contig sequence total: 5.935 MB 0.016% gap
Main genome scaffold N/L50: 5/291.264 KB
Main genome contig N/L50: 7/277.876 KB
Main genome scaffold N/L90: 16/141.784 KB
Main genome contig N/L90: 21/106.371 KB
Max scaffold length: 1.081 MB
Max contig length: 862.609 KB
Number of scaffolds > 50 KB: 21
% main genome in scaffolds > 50 KB: 97.73%
Minimum Number Number Total Total Scaffold
Scaffold of of Scaffold Contig Contig
Length Scaffolds Contigs Length Length Coverage
-------- -------------- -------------- -------------- -------------- --------
All 116 126 5,935,480 5,934,503 99.98%
100 116 126 5,935,480 5,934,503 99.98%
250 105 115 5,933,465 5,932,488 99.98%
500 58 68 5,918,943 5,917,966 99.98%
1 KB 38 48 5,906,055 5,905,078 99.98%
2.5 KB 32 42 5,896,271 5,895,294 99.98%
5 KB 26 34 5,874,307 5,873,525 99.99%
10 KB 24 32 5,860,741 5,859,959 99.99%
25 KB 22 30 5,830,221 5,829,439 99.99%
50 KB 21 29 5,800,895 5,800,113 99.99%
100 KB 18 25 5,594,616 5,593,931 99.99%
250 KB 8 13 3,843,092 3,842,607 99.99%
500 KB 2 5 1,898,417 1,898,127 99.98%
1 MB 1 2 1,081,451 1,081,351 99.99%
## 5. estimate insert size
~/work/bbmap/bbmap.sh ref=/spades_NG_1783_bbduk_norm_denovo_cf_v4/contigs.fasta in1=NG_1783_R1_bbduk.fastq.gz in2=NG_1783_R2_bbduk.fastq.gz ihist=ihist_mapping.txt out=mapped.sam maxindel=200000
Pairing data: pct pairs num pairs pct bases num bases
mated pairs: 98.6907% 8171939 98.6898% 2464559742
bad pairs: 0.9356% 77471 0.9363% 23383188
insert size avg: 422.12
insert 25th %: 315.00
insert median: 389.00
insert 75th %: 478.00
insert std dev: 588.63
insert mode: 359
##########################################################
## 4. blast against ref genome and use contigs as query ##
# 4.1 SCSIO 6842 BLAST
makeblastdb -in GCF_001482385.1_ASM148238v1_genomic.fna -out GCF_001482385.1_ASM148238v1_genomic -dbtype 'nucl' -hash_index
blastn -query spades_NG_1783_bbduk_norm_denovo_cf_v4/scaffolds.1000.fasta -max_target_seqs 1 -max_hsps 1 -evalue 10 -outfmt 6 -out spades_NG_1783_bbduk_norm_denovo_cf_v4_contigs.txt -db SCSIO6842blast/GCF_001482385.1_ASM148238v1_genomic -num_threads 8