Error: Add anchor information into re-alignment BAM file

[samarth51]

Hi
I have BWA bam files. I tried to run ./Socrates realignment step but got an error message:
Add anchor information into re-alignment BAM file

Command given my me is:
./Socrates inputbam(generated in first step of socrates) outputbam

Any help will be help full.
Thanks

[jibsch]

Try running the program from start to finish by calling Socrates all and filling in the necessary parameters.
Please let me know if that gets you there.

[samarth51]

Hi,
I tried running with Socrates all as well using command:
./Socrates all InputBAM
This command gives me a message on screen:

"Bowtie2 DB is required to perform soft-clip realignment. Please specify this parameter with --bowtie2_db "

In help section this parameter is defined as:
bowtie2_db BOWTIE2_DB -- Prefix of Bowtie2 indexed database for sample (default: None)
What "None" is for here?? It creates confusion.

Another thing is I have BWA generated BAM files so in that case what will be the value of "bowtie2_db" parameter?? Do i need bowtie2 generated BAM only to use Socrates??

Need suggestions on this.

[jibsch]

The "None" default is supposed to indicate that this parameter is required
as depends on the data.
It is fine to use BWA alignments, but the re-alignment is to be done with
bowtie2 (at least for the time being).
Create a bowtie2 index with the bowtie2-build command. Provide the prefix
of the resulting set of files (not including ".") as the bowtie2_db
parameter.

On 6 July 2015 at 18:33, Samarth Kulshrestha [email protected]
wrote:

Hi,
I tried running with Socrates all as well using command:
./Socrates all InputBAM
This command gives me a message on screen:

"Bowtie2 DB is required to perform soft-clip realignment. Please specify
this parameter with --bowtie2_db "

In help section this parameter is defined as:
bowtie2_db BOWTIE2_DB -- Prefix of Bowtie2 indexed database for sample
(default: None)
What "None" is for here?? It creates confusion.

Another thing is I have BWA generated BAM files so in that case what will
be the value of "bowtie2_db" parameter?? Do i need bowtie2 generated BAM
only to use Socrates??

Need suggestions on this.

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#15 (comment).

[samarth51]

Hi,
Thanks for valuable suggestions. Everything is going good so far.
I have one another query. I have paired samples (Tumor vs Normal) so what parameter i need to adjust for paired samples?

Thanks

[jibsch]

Glad that worked.
For tumour vs normal: Run both samples independently first. Then use the
Socrates annotate module and specify the normal results file (paired) after
"--normal".

On 8 July 2015 at 16:08, Samarth Kulshrestha [email protected]
wrote:

Hi,
Thanks for valuable suggestions. Everything is going good so far.
I have one another query. I have paired samples (Tumor vs Normal) so what
parameter i need to adjust for paired samples?

Thanks

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#15 (comment).

[samarth51]

Hi,
I ran tumor and normal samples separately, i got raw output for both the samples and all went well so far. Now to find out somatic SVs what parameters needs to be used ?

./Socrates annotate --features raw_tumor -- normal raw_blood
Is this the right way ??

Thanks

[jibsch]

Almost:
./Socrates annotate --normal raw_blood raw_tumor

On 14 July 2015 at 17:10, Samarth Kulshrestha [email protected]
wrote:

Hi,
I ran tumor and normal samples separately, i got raw output for both the
samples and all went well so far. Now to find out somatic SVs what
parameters needs to be used ?

./Socrates annotate --features raw_tumor -- normal raw_blood
Is this the right way ??

Thanks

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#15 (comment).

[samarth51]

Hi.
I performed annotation and get SV breakpoints. But i have few doubts regarding output.

1. Accoring to SV typing criterion (assuming C1 realign pos < C1 anchor pos) DELETION would be if : C1_realign_dir + & C1_anchor_dir -
   I applied the mentioned criterion and get few breakpoints on different chromosome in case of Deletion.

chr1:9849740 + CCTTTAAGCTCTATTGGACTTGATATGGTTAGTTTTAAAAAGA chr2:130489048 - TAAAGAACAATAAAGGCCAGGCACTGTGGCTCATACCTGTAATCCCAGCACTTTGGG 1 43 0 0 0 39.0 chr2:130489052 - GAACAATAAAGGCCAGGCACTGTGGCTCA chr1:9849744 + TTTACCTTTAAGCTCTATTGGACTTGATATGGTTAGTTTTAAAAAGAGTTGTTAGCTTTTAGAGATGTATG 1 29 00 0 38.0 Micro-homology: 4bp homology found! (TAAA)

This read has C1_realign chr1:9849740 and C1_anchor chr2:130489048 . Why this happened for a DELETION event??

2. I also have SV breakpoints in output where C1 realign pos > C1 anchor pos. So how to deal with this kind of breakpoints?

Please give some suggestions for the problem.

[jibsch]

Hi,
Socrates calls fusions between two coordinates. The orientation of the
breakpoints (+/-) determines what kind of event it is. In case 1) it is
indeed a deletion signature: coordinate 1 < coordinate 2, and orientations
+, -. Case 2) occurs, if realignment was successful on one side of the
fusion only. You can treat them the same way: if the smaller coordinate has
a +, the second a -, it's the deletion signature, -+ for tandem
duplication, ++, -- for inversions types. More complex events contain more
than one fusion and are not as easily identifiable.
Cheers,
Jan

On 21 July 2015 at 17:59, Samarth Kulshrestha [email protected]
wrote:

Hi.
I performed annotation and get SV breakpoints. But i have few doubts
regarding output.

1. Accoring to SV typing criterion (assuming C1 realign pos < C1 anchor
   pos) DELETION would be if : C1_realign_dir + & C1_anchor_dir -

I applied the mentioned criterion and get few breakpoints on different
chromosome in case of Deletion.

chr1:9849740 + CCTTTAAGCTCTATTGGACTTGATATGGTTAGTTTTAAAAAGA chr2:130489048

• TAAAGAACAATAAAGGCCAGGCACTGTGGCTCATACCTGTAATCCCAGCACTTTGGG 1 43 0 0 0 39.0
  chr2:130489052 - GAACAATAAAGGCCAGGCACTGTGGCTCA chr1:9849744 +
  TTTACCTTTAAGCTCTATTGGACTTGATATGGTTAGTTTTAAAAAGAGTTGTTAGCTTTTAGAGATGTATG 1
  29 00 0 38.0 Micro-homology: 4bp homology found! (TAAA)

This read has C1_realign chr1:9849740 and C1_anchor chr2:130489048 . Why
this happened for a DELETION event??

2. I also have SV breakpoints in output where C1 realign pos > C1 anchor
   pos. So how to deal with this kind of breakpoints?

Please give some suggestions for the problem.

—
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#15 (comment).

[samarth51]

Hi
I used --repeatmask (UCSC repeat masker track) for annotation purpose. I get output column with name "repeat1" "repeat2" but do not get any value for these columns. So what are the expected values or output for those columns if i use repeat masker track ??
UCSC repeat masker track format
chr1 16777160 16777470 AluSp 2147 +
chr1 25165800 25166089 AluY 2626 -

[jibsch]

That's interesting. Can you try again by using --features instead of
--repeatmask?
Otherwise, are the chromosome names in the Socrates output the same as in
the annotation?

On 5 August 2015 at 18:00, Samarth Kulshrestha [email protected]
wrote:

Hi
I used --repeatmask (UCSC repeat masker track) for annotation purpose. I
get output column with name "repeat1" "repeat2" but do not get any value
for these columns. So what are the expected values or output for those
columns if i use repeat masker track ??
UCSC repeat masker track format
chr1 16777160 16777470 AluSp 2147 +
chr1 25165800 25166089 AluY 2626 -

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#15 (comment).

[samarth51]

I tried with --features( gene coordinates) this give me "feature1" and "feature2" with gene names but --repeatmask parameter does not output anything. Yes chromosome names in the output and annotation are same... Any suggestion for repeatmask??

[jibsch]

Sorry, I meant using --features and the repeat track. Does that give you
repeat annotation, or does the problem persist?

On 5 August 2015 at 23:16, Samarth Kulshrestha [email protected]
wrote:

I tried with --features( gene coordinates) this give me "feature1" and
"feature2" with gene names but --repeatmask parameter does not output
anything. Yes chromosome names in the output and annotation are same... Any
suggestion for repeatmask??

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#15 (comment).

[samarth51]

Hi,
I tried all the possible combinations of parameters. I also ran a combination of --feature (UCSS_repeatmask) and gets repeat annotation (output pasted below).
C1_realign C1_realign_dir C1_realign_consensus C1_anchor C1_anchor_dir C1_anchor_consensus C1_long_support C1_long_support_bases C1_short_support C1_short_support_bases C1_short_support_max_len C1_avg_realign_mapq C2_realign C2_realign_dir C2_realign_consensus C2_anchor C2_anchor_dir C2_anchor_consensus C2_long_support C2_long_support_bases C2_short_support C2_short_support_bases C2_short_support_max_len C2_avg_realign_mapq BP_condition normal feature1 feature2

chr1:821605 - GCCCTTTGGCAGAGCAGGTGTGCTGTGCTGTGCTGATCCCCGGGAGTC chr1:821634 + CAGCACAGCACACCTGCTCTGCCAAAGGGCAGCCAGACTGCTTCTTTAAGCAGTTCCTGATCTTGTTT 11 443 15 215 23 37.363636 chr1:821634 + CAGCACAGCACACCTGCTCTGCCAAAGGGCAGCCAGACTGCTTCTTTA chr1:821605 - GCCCTTTGGCAGAGCAGGTGTGCTGTGCTGTGCTGATCCCCGGGAGTCTCCAGAGCC
AGCAGGCTGGA 13 519 11 100 15 29.692308 Blunt-end joining normal L1PA7 L1PA7.

When i provide repeatmasker file for --repeatmak it does not work but when i provide the same repeatmasker file for --feature parameter it works (output pasted above).