Bowtie2 aligner

Question

[priya.bmg]

Hello

I am trying to learn Bowtie and I couldn't figure out the error.

First, I downloaded GRCH38 build from NCBI assembly (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.39) and using the command bowtie2-build, built indexes of the reference sequence. Got four index bt2 files Then downloaded fasta sequence from NCBI SRA (http://ftp.sra.ebi.ac.uk/vol1/run/ERR235/ERR2356727/24_1.fastq.gz) (http://ftp.sra.ebi.ac.uk/vol1/run/ERR235/ERR2356727/24_2.fastq.gz)

Then aligned with reference sequence using the command: bowtie2 -x GCF_000001405.26_GRCh38_genomic.fna.fna -U 24_1.fastq,24_2.fastq -S eg1.sam

I get the following error: "GCF_000001405.26_GRCh38_genomic.fna.fna" does not exist or is not a Bowtie 2 index

I used the bowtie2 commands to align the sequence but still getting an error. Can someone help with the error here? Is there a way to make sure that I have Bowtie 2 index files

Thanks

Priya

[GenoMax]

Can you provide the command you used to build your bowtie2 indexes and also provide a listing of ls -lh GCF*. Are you sure the indexes were properly made without any errors at the end of that process?

[priya.bmg]

Blockquote

bowtie2-build GCF_000001405.26_GRCh38_genomic.fna bt2_index ## Build indexes

Blockquote

ls -lh GCT*

>  rw-r--r-- 1 thirun0000 cichon 3.1G Jul 27 10:06
> -rw-r--r-- 1 thirun0000 cichon  16K Jul 27 10:09 GCF_000001405.26_GRCh38_genomic.fna.index.1.bt2
> -rw-r--r-- 1 thirun0000 cichon    0 Jul 27 10:08 GCF_000001405.26_GRCh38_genomic.fna.index.2.bt2
> -rw-r--r-- 1 thirun0000 cichon  15K Jul 27 10:08 GCF_000001405.26_GRCh38_genomic.fna.index.3.bt2
> -rw-r--r-- 1 thirun0000 cichon 728M Jul 27 10:08 GCF_000001405.26_GRCh38_genomic.fna.index.4.bt2

[GenoMax]

Possible multiple issues. Do you have a file with no readable name that is 3.1G? It looks like your bowtie-build command may have failed part-way. You should capture the stdout/stderr to capture those messages. You are missing rev files as you have already discovered.

bowtie2-build GCF_000001405.26_GRCh38_genomic.fna  index_base_name (use a name you like) > log.out 2>log.error 

Then look at the log files produced.

[priya.bmg]

The 3.1 GB is the reference file downloaded from NCBI assembly

log.error

Building a SMALL index

log.out

Settings:
  Output files: "index_base_name_alignment.*.bt2"
  Line rate: 6 (line is 64 bytes)
  Lines per side: 1 (side is 64 bytes)
  Offset rate: 4 (one in 16)
  FTable chars: 10
  Strings: unpacked
  Max bucket size: default
  Max bucket size, sqrt multiplier: default
  Max bucket size, len divisor: 4
  Difference-cover sample period: 1024
  Endianness: little
  Actual local endianness: little
  Sanity checking: disabled
  Assertions: disabled
  Random seed: 0
  Sizeofs: void*:8, int:4, long:8, size_t:8
Input files DNA, FASTA:
  GCF_000001405.26_GRCh38_genomic.fna
Reading reference sizes
  Time reading reference sizes: 00:00:24
Calculating joined length
Writing header
Reserving space for joined string
Joining reference sequences
  Time to join reference sequences: 00:00:16
bmax according to bmaxDivN setting: 762328945
Using parameters --bmax 571746709 --dcv 1024
  Doing ahead-of-time memory usage test
  Passed!  Constructing with these parameters: --bmax 571746709 --dcv 1024
Constructing suffix-array element generator
Building DifferenceCoverSample
  Building sPrime
  Building sPrimeOrder
  V-Sorting samples
  V-Sorting samples time: 00:01:29
  Allocating rank array
  Ranking v-sort output
  Ranking v-sort output time: 00:00:22
  Invoking Larsson-Sadakane on ranks
  Invoking Larsson-Sadakane on ranks time: 00:00:46
  Sanity-checking and returning
Building samples
Reserving space for 12 sample suffixes
Generating random suffixes
QSorting 12 sample offsets, eliminating duplicates
QSorting sample offsets, eliminating duplicates time: 00:00:00
Multikey QSorting 12 samples
  (Using difference cover)
  Multikey QSorting samples time: 00:00:00
Calculating bucket sizes
Splitting and merging
  Splitting and merging time: 00:00:00
Avg bucket size: 3.04932e+09 (target: 571746708)
Converting suffix-array elements to index image
Allocating ftab, absorbFtab
Entering Ebwt loop
Getting block 1 of 1
  No samples; assembling all-inclusive block

[GenoMax]

Is that all you get? At least in this part there is no error. Perhaps the process is not yet complete? Assuming you re-ran the command just now it should not take < 5 min to complete the indexing.

[priya.bmg]

Yes. Is there a way to get pre-built indexes for bowtie2?

[GenoMax]

You can get pre-built indexes from Bowtie SF page. Look in the right column as you scroll down.

[priya.bmg]

I have downloaded fastq files of the reference sequence which created a problem in building indexes. fasta files have to used for building indexes (http://seqanswers.com/forums/showthread.php?t=14673). I downloaded prebuilt indexes for GRCh38 from ftp://ftp.ccb.jhu.edu/pub/data/bowtie2_indexes/grch38_1kgmaj_bt2.zip and I had no issues with alignment. Thank you everyone for their suggestions, it was very helpful

Answer 1

[Jianyu]

The prefix of index should be "GCF_000001405.26_GRCh38_genomic.fna.index" instead of "GCF_000001405.26_GRCh38_genomic.fna.fna"

[priya.bmg]

Now I get a different error:

 > Could not open index file GCF_000001405.26_GRCh38_genomic.fna.index.rev.1.bt2
 > Could not open index file GCF_000001405.26_GRCh38_genomic.fna.index.rev.2.bt2 
    (ERR): bowtie2-align died with signal 11 (SEGV)

I have no reverse index files

[Jianyu]

Could you show us the log information of your "bowtie2-build" command? It seems "GCF_000001405.26_GRCh38_genomic.fna.index.2.bt2" is empty, which suggests bowtie2-build might failed

[priya.bmg]

Please find the log file posted above. Thanks