Question

Rockhopper Reference Genome Error

0

Entering edit mode

5 weeks ago

ATS • 0

Hello,

I'm trying to use Rockhopper in the command line to analyze some RNA-seq results. I can get the de novo transcript assembly working just fine, but when I attempt a reference based analysis it begins to throw errors.

I am running the following line on a cluster (only including one RNA-seq replicate for clarity):

java -Xmx1200m -cp Rockhopper.jar Rockhopper -g RockhopperRef/ Sample1_1_sequence.fastq%Sample1_2_sequence.fastq -o RefAlignment

where "RockhopperRef" is a directory with a single set of genome.fna, genes.ptt, and genes.rnt files.

The error I get is:

Exception in thread "main" java.util.NoSuchElementException: No line found
    at java.util.Scanner.nextLine(Scanner.java:1540)
    at Genome.readInGenes(Genome.java:309)
    at Genome.<init>(Genome.java:74)
    at Rockhopper.<init>(Rockhopper.java:99)
    at Rockhopper.main(Rockhopper.java:908)

I've gone into the rockhopper.jar code and looked at the lines that are being flagged. They all seem to be related to reading/referencing the .ptt file, but I cannot figure out why it's unhappy. Here is the format of my .ptt file, for reference:

complete sequence - 0..1300386
1454 proteins
Location    Strand  Length  PID Gene    Synonym Code    COG Product
222..659    -   145 -   hemJ    MC1_RS00005 -   -   protoporphyrinogen oxidase HemJ
671..1711   -   346 -   hemH    MC1_RS00010 -   -   ferrochelatase
1668..2708  -   346 -   hemE    MC1_RS00015 -   -   uroporphyrinogen decarboxylase

Any ideas on what the problem could be? Alternatively, is there a way to call a reference genome directly from GenBank while using the command line version of Rockhopper, similar to what's available in the GUI? The issue seems to be something with my genome directory, so if I could call the genome directly from Genbank, that might bypass this issue.

Thank you for taking the time to read my question! This is driving me nuts, and I'm so appreciative of any advice.

Rockhopper • 366 views

ADD COMMENT • link 5 weeks ago by ATS • 0

0

Entering edit mode

Genome counting starts from 1, not 0. That may or may not be the reason for the error.

This is now my .ptt file looks:

Escherichia coli str. K-12 substr. W3110, complete genome - 1..4646332
4213 proteins
Location        Strand  Length  PID     Gene    Synonym Code    COG     Product
190..255        +       21      388476124       thrL    Y75_p0001       -       -       thr operon leader peptide
337..2799       +       820     388476125       thrA    Y75_p0002       -       -       fused aspartokinase I and homoserine dehydrogenase I
2801..3733      +       310     388476126       thrB    Y75_p0003       -       -       homoserine kinase
3734..5020      +       428     388476127       thrC    Y75_p0004       -       -       threonine synthase
5234..5530      +       98      388476128       yaaX    Y75_p0005       -       -       hypothetical protein
5683..6459      -       258     388476129       yaaA    Y75_p0006       -       -       hypothetical protein
6529..7959      -       476     388476130       yaaJ    Y75_p0007       -       -       transporter