Transcript 12. Pre processing Metagenomic Datasets

(Meta)genomic dataset
preprocessing
Konstantinos Mavrommatis
[email protected]
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The underlying question(s) ….
• What …#$#%*$ / <3 <3… is happening on my
dataset after I submit it to IMG/ER?
• Why don’t I see the results immediately on
IMG?
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Dataset processing
Sample preparation
High throughput sequencing
Assemble reads
Analysis
Feature prediction
QC
Functional annotation
and comparative analysis
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Outline
• Data preprocessing
• Annotation
• Time considerations
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Dataset pre-processing
(v 3.0a)
Submitted file
Submitted file
Submitted file
Assembled contigs
454 reads
Illumina reads
Fasta/fast
q
File QC.
Check character set and contig name. Remove trailing Ns.
Trimming.
Trimming.
Q=20
Q=13
Fasta
Low complexity.
Size of 80 bp
Dereplication.
Prefix = 5, identity 95%,
Clustering.
100% identity
File for gene calling
fasta
Dataset pre-processing
Quality trimming
Courtesy Alex Copeland
http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/
Remove sequences from the ends of the reads.
lucy for 454 datasets.
Illumina (longest high quality string)
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Dataset pre-processing
Low complexity filter
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using dust (NCBI)
-Remove sequences with less than 80
informative bases
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Dataset pre-processing
Dereplication
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Dataset pre-processing
Sequence dereplication
atcccat
atc-cat
atcccat
atcccat
atcccat
gctacat
gctncat
gctacat
gctacat
Not
dereplicated
using uclust
-95% identity (global alignment).
-Identical prefix (5nt)
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Pre-processing in a nutshell
• Quality trimming
• Low complexity
trimming
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• Dereplication
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Outline
• Data preprocessing
• Annotation
• Time considerations
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Dataset processing
Feature prediction pipeline (v 3.0a)
File for gene calling
fasta
Unassembled reads + assembled contigs
CRISPR detection.
crt / pilercr
RNA detection.
tRNAscan / hmmer / Blast / (isolates:Rfam)
CDS detection.
Isolates: prodigal
Metagenomes: varies
Conflict resolution
Concatenation of all results.
Creation of final output file
File for IMG
IMG
Genomes
• Prodigal to predict CDS
• tRNA scan to predict tRNAs
• In house models for rRNA
• Infernal for ncRNA
• CRISPR detection
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Gene calling on metagenomes is
harder and error prone
Unassembled sequences
•small size,
•quality problems,
•large number
Assembled sequences frequently
•contain errors,
•low quality regions,
•fragmented genes.
Gene calling is not accurate.
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Dataset processing
Feature prediction
Simulated datasets:
a.Using fake reads extracted from finished genomes (Perfect sequences)
b.Using real reads that have been used to assemble finished genomes (Real
errors).
isolate
CORRECT
metagenome
MISSED
NEW
WRONG
Available methods:
Ab initio: FragGeneScan,
Metagene, MetaGeneMark,
Prodigal.
Similarity based: Blastx,
USEARCH.
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Trimming
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454 Ti(no errors)
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454Ti(with errors)
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Illumina 115 bp
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Contigs
frameshift
Wrong prediction
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Genomes
• Weighted gene callers to
predict CDS
• tRNAscan & blastn to predict
tRNAs
• In house models for rRNA
• Infernal for ncRNA
• CRISPR detection
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Functional annotation
• COG/KOG
• Pfam/TIGRfam
• Usearch vs reference
– KO terms/EC #
– Phylogenetic distribution
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Outline
• Data preprocessing
• Annotation
• Time considerations
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Processing time(metagenomes)
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Total submissions
Processing time
336
2.45 days (annotation)
Data size (bp)
174,719,855 (average)
Processing time(isolates)
Total submissions
3630
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Processing time
10 hours(annotation)
12 days (integration)
Data size (bp)
1,658,242 (average)
4,114,099,773
(total)
The underlying question ….
Time for your questions
• What …#$#%*$ / <3 <3… is happening on my
dataset after I submit it to IMG/ER?
• Patience is a virtue :
It takes a lot of computations… and there are
many datasets to be processed.
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