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Metagenomics - Remove Host Sequences

This document provides instructions for removing host sequences from metagenomic sequencing data in silico using Bowtie2 and SAMtools. There are two main approaches: 1) Using only Bowtie2 with the --un-conc option to get unmapped reads in separate files. 2) Using Bowtie2 to map to the host genome and write all reads to a BAM file, then using SAMtools flags to extract unmapped reads and write them to separate FASTQ files. The goal is to generate host-filtered FASTQ files for downstream analysis of microbial sequences.

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320 views

Metagenomics - Remove Host Sequences

This document provides instructions for removing host sequences from metagenomic sequencing data in silico using Bowtie2 and SAMtools. There are two main approaches: 1) Using only Bowtie2 with the --un-conc option to get unmapped reads in separate files. 2) Using Bowtie2 to map to the host genome and write all reads to a BAM file, then using SAMtools flags to extract unmapped reads and write them to separate FASTQ files. The goal is to generate host-filtered FASTQ files for downstream analysis of microbial sequences.

Uploaded by

emilio
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Metageno…

Remove host
sequences
Metagenomics

Tools

16S tools

Assembly

BLAST

Bowtie2 In silico removal of host sequences


Genome removing host (contamination) sequences in order to analyze remaining (bacterial) sequences

Pathogen screening

1) Using bowtie2 option: --un-conc


Phylogenetic tree

SAMtools

Sequence data
quick solution to get the paired reads that do not map to the host reference genome (both reads unmapped).
Shotgun sequencing
# download ready to use bowtie2 database of human host genome GRCh38 (hg38)
Alignment wget https://genome-idx.s3.amazonaws.com/bt/GRCh38_noalt_as.zip
unzip GRCh38_noalt_as.zip
Data

Fastq file format

NCBI SRA file # run bowtie2 mapping (using --un-conc-gz to get gzip compressed output files; 8 processors)
format bowtie2 -p 8 -x GRCh38_noalt_as -1 SAMPLE_R1.fastq.gz -2 SAMPLE_R2.fastq.gz --un-conc-gz
SAMPLE_host_removed > SAMPLE_mapped_and_unmapped.sam
Quality control

Remove host http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#output-options


sequences
# bowtie2 results (gz files without gz ending)
Remove too short ls
reads from fastq
files
SAMPLE_host_removed.1
Ubuntu/Linux server
SAMPLE_host_removed.2

°°°

# rename host-sequence free samples


mv SAMPLE_host_removed.1 SAMPLE_host_removed_R1.fastq.gz
mv SAMPLE_host_removed.2 SAMPLE_host_removed_R2.fastq.gz

Option --un-conc shows results like samtools options -F 2 (excluding reads "mapped in proper pair").

Paired reads that do not map both to the host sequence might still be included in the "host removed" output.

For better control about read filtering options, see workflow below.

If multi-processor option -p is used, output reads might have a different order compared to input files.

Use option --reorder to keep the original read order.

(read order refers to .sam output but might effect also host-removed read output files .1 .2)

http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#performance-options

2) Using bowtie2 together with


samtools
complex solution that gives better control over the rejected reads by using SAM-flags

How to filter out host reads from paired-end fastq files?

a) bowtie2 mapping against host genome: write all (mapped and unmapped) reads to a single .bam file

b) samtools view: use filter-flags to extract unmapped reads

c) samtools fastq: split paired-end reads into separated R1 and R2 fastq files

a) bowtie2 mapping against host sequence


1) create or download ready to use bowtie2 index databases (host_DB)

# create bowtie2 database using a reference genome (fasta file)


bowtie2-build host_genome_sequence.fasta host_DB

→ Download reference genomes of common hosts (human, mouse, ..)

→ How to create a bowtie2 index database

# download ready to use bowtie2 database of human genome GRCh38 (hg38)

wget https://genome-idx.s3.amazonaws.com/bt/GRCh38_noalt_as.zip
unzip GRCh38_noalt_as.zip

# move all files into your working directory (or into your predefined $BOWTIE2_INDEXES location)

# use "GRCh38_noalt_as" for your bowtie2 database name (instead of "host_DB")

→ see all available bowtie2 databases (host species list is shown on the right)

2) bowtie2 mapping against host sequence database, keep both aligned and unaligned reads (paired-end reads)
bowtie2 -p 8 -x host_DB -1 SAMPLE_R1.fastq.gz -2 SAMPLE_R2.fastq.gz -S
SAMPLE_mapped_and_unmapped.sam

3) convert file .sam to .bam


samtools view -bS SAMPLE_mapped_and_unmapped.sam > SAMPLE_mapped_and_unmapped.bam

b) filter required unmapped reads


# SAMtools SAM-flag filter: get unmapped pairs (both reads R1 and R2 unmapped)

samtools view -b -f 12 -F 256 SAMPLE_mapped_and_unmapped.bam > SAMPLE_bothReadsUnmapped.bam


-f 12 Extract only (-f) alignments with both reads unmapped: <read unmapped><mate unmapped>

-F 256 Do not(-F) extract alignments which are: <not primary alignment>

see meaning of SAM-flags

c) split paired-end reads into separated fastq files .._R1 .._R2


# sort bam file by read name (-n) to have paired reads next to each other (2 parallel threads, each using up to 5G memory)

samtools sort -n -m 5G -@ 2 SAMPLE_bothReadsUnmapped.bam -o SAMPLE_bothReadsUnmapped_sorted.bam


samtools fastq -@ 8 SAMPLE_bothReadsUnmapped_sorted.bam \
-1 SAMPLE_host_removed_R1.fastq.gz \
-2 SAMPLE_host_removed_R2.fastq.gz \
-0 /dev/null -s /dev/null -n

http://www.htslib.org/doc/samtools-fasta.html

see other options for → converting .bam to .fastq

Result
Two files of paired-end reads, containing non-host sequences

SAMPLE_host_removed_R1.fastq.gz
SAMPLE_host_removed_R2.fastq.gz

See also:

Removel of ribosomal RNA


To in silico remove rRNA, see software tool → SortMeRNA

Author: Matthias Scholz


https://twitter.com/panphlan

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